Expression of granular starch hydrolyzing enzymes in Trichoderma and process for producing glucose from granular starch substrates

ABSTRACT

The present invention relates to filamentous fungal host cells and particularly  Trichoderma  host cells useful for the production of heterologous granular starch hydrolyzing enzymes having glucoamylase activity (GSHE). Further the invention relates to a method for producing a glucose syrup comprising contacting a granular starch slurry obtained from a granular starch substrate simultaneously with an alpha amylase and a GSHE at a temperature equal to or below the gelatinization temperature of the granular starch to obtain a composition of a glucose syrup.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application claiming priority to U.S.patent application Ser. No. 11/875,279, filed Oct. 19, 2007, whichclaims priority to U.S. patent application Ser. No. 10/991,654, filedNov. 18, 2004, now issued as U.S. Pat. No. 7,303,899, issued Dec. 4,2007, claiming priority to U.S. Provisional Application No. 60/524,279,filed Nov. 21, 2003, U.S. Provisional Application No. 60/531,953, filedDec. 22, 2003, and U.S. Provisional Application No. 60/566,358, filedApr. 28, 2004. The disclosures of the priority applications areincorporated by reference in their entirety.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with C.F.R.§1.52(e), is incorporated herein by reference. The sequence listing textfile submitted via EFS contains the file “GC824D1C1_SeqListing.TXT”,created on Feb. 24, 2012, which is 39,952 bytes in size.

FIELD OF THE INVENTION

The present invention relates to filamentous fungal host cells usefulfor the expression of heterologous granular starch hydrolyzing enzymeshaving glucoamylase activity (GSHE). The invention further relates tothe use of the GSHE in methods for producing glucose syrup and other endproducts from granular starch substrates comprising contacting agranular starch substrate, at or below the gelatinization temperature ofthe granular starch, simultaneously with a starch liquefying amylase anda GSHE. The invention further relates to enzyme compositions comprisingthe GSHE and starch liquefying amylase.

BACKGROUND OF THE INVENTION

Industrial fermentation predominately uses glucose as a feedstock forthe production of a multitude of proteins, enzymes, amino acids,alcohols, organic acids, pharmaceuticals and other chemicals. In manyapplications, the glucose is produced from the enzymatic conversion ofcarbon substrates such as biomass and starch. Starch, which isabundantly found in green plants, accumulates as microscopic granulesvarying in diameter from 0.5 to 175 microns. The partial crystallinenature of these starch granules imparts insolubility in cold water. As aresult, the solubilization of starch granules in water requires atremendous amount of heat energy to disrupt the crystalline structure ofthe granule resulting in the solubilization of partially hydrolyzedstarch. Numerous solubilization processes have been employed and theseinclude direct and indirect heating systems, such as direct heating bysteam injection. (See for example, STARCH CHEMISTRY AND TECHNOLOGY, edsR. L. Whistler et al., 2^(nd) Ed., 1984 Academic Press Inc., Orlando,Fla.; STARCH CONVERSION TECHNOLOGY, Eds. G. M. A. Van Beynum et al.,Food Science and Technology Series, Marcel Dekker Inc., NY; and THEALCOHOL TEXTBOOK, 3^(rd) Ed., Eds. K. Jacques, T. P. Lyons and D. R.Kelsall, 1999, Nottingham University Press, UK).

In general, two enzyme steps have been used for the hydrolysis of starchto glucose. The first step is a liquefaction step, and the second stepis a saccharification step. In the liquefaction step, the insolublestarch granules are slurried in water, gelatinized with heat andhydrolyzed by a thermostable alpha amylase (EC.3.2.1.1, alpha(1-4)-glucan glucanohydrolase) in the presence of added calcium toproduce a mash of dextrins. The resulting mash is generally cooled toabout 60 to 65° C. In the saccharification step, the soluble dextrins(sugars) are further hydrolyzed to dextrose (glucose) by an enzymehaving glucoamylase (EC 3.2.1.3,alpha (1,4)-glucan glucohydrolase)activity. Glucose may then be used as an end product or used as aprecursor to be converted into other commercially important desired endproducts, such as fructose, sorbitol, ethanol, lactic acid, ascorbicacid (ASA) intermediates and 1,3 propanediol.

In the late 1950s, glucoamylases derived from Aspergillus niger werecommercialized, and these enzymes significantly improved the conversionof starch to glucose. Another significant improvement occurred in the1970s. A thermostable alpha amylase having improved thermostability, pHstability and lower calcium dependency was derived and commercializedfrom Bacillus licheniformis (U.S. Pat. No. 3,912,590).

Further industrial processes have been adopted by the starch sweetenerindustry for the enzyme liquefaction process (U.S. Pat. No. 5,322,778).Some of these processes include, a low temperature process (105-110° C.for 5-8 min) with lower steam requirements and a high temperatureprocess (148° C.+/−5° C. for 8-10 sec), which improves gelatinization ofthe starch granules resulting in improved filtration characteristics andquality of the liquefied starch substrate (Shetty, et al., (1988) CerealFoods World 33:929-934).

While enzyme starch liquefaction processes are well established,improvements with respect to yield loss, processing costs, energyconsumption, pH adjustments, temperature thresholds, calcium requirementand the levels of retrograded starch would be desirable. In particular,it is well known that residual alpha amylase from the liquefaction step,under saccharification conditions, has an adverse effect on processefficiency and that the residual alpha amylase must be inactivated priorto saccharification by glucoamylases. Inactivation is generallyaccomplished by lowering the pH of the liquefied starch to pH 4.2 to 4.5at 95° C. Another disadvantage of liquefaction processes is the alkalineisomerization of reducing groups. Alkaline isomerization results in theformation of a disaccharide, maltulose(4-alpha-D-glucopyranosyl-D-fructose). Maltulose lowers the glucoseyield because it is resistant to hydrolysis by glucoamylases and alphaamylases. Further, it is difficult to control the formation of reversionreaction products catalyzed by active glucoamylases at high glucoseconcentration. Glucoamylases from Aspergillus niger are generallythermostable under the typical saccharification conditions. Therefore, asubstantial amount of the glucoamylase activity may remain after thesaccharification reaction. Solutions to some of the problems asdiscussed herein have been suggested by various researchers.

For example, Leach et al (U.S. Pat. No. 4,113,509 and U.S. Pat. No.3,922,196) disclose a process for converting granular starch (refined)into soluble hydrolyzate by incubating the granular starch withbacterial alpha amylase at a temperature below the starch gelatinizationtemperature. Beta amylase was then used for hydrolysis to produce highmaltose syrup.

European Patent Application No. 0350737 A2 discloses a process forproducing maltose syrup by hydrolyzing a granular (purified) starch fromcorn, wheat, potato and sweet potato at 60° C. without the conventionalliquefaction step (gelatinization followed by liquefaction at hightemperature) using an alpha amylase from Bacillus stearothermophilus.

A multi-step process to convert granular (raw) starch to glucose using aglucoamylase demonstrating raw starch hydrolyzing capability has beenpreviously described (U.S. Pat. No. 4,618,579). However, only 60% of thestarch was hydrolyzed, which then resulted in an extensive recyclingprocess.

Not only would it be advantageous to improved upon conventionalprocesses for granular starch conversion, but also it would be desirableto provide processes resulting in increased expression and production ofthe enzymes used therefore. For example, glucoamylases having granularstarch hydrolyzing activity with improved characteristics such asincreased specific activity, different pH ranges and/or different levelsof glycosylation may be particularly advantageous for use in industrialstarch conversion. The present invention not only meets some of theseneeds but also results in an increase in the efficiency of producingvarious end products obtained from starch hydrolysis.

BRIEF SUMMARY OF THE INVENTION

One embodiment of the present invention concerns a one-step process forconverting granular starch to glucose by hydrolyzing granular starch ator below the gelatinization temperature of the granular starchsubstrate, by simultaneously contacting the ungelatinized starchsubstrate with an endo-acting alpha amylase and a saccharifying enzymehaving glucoamylase activity, and more specifically having granularstarch hydrolyzing activity.

The present invention finds utility and improvement over prior artprocesses in at least one of the following ways a) both the alphaamylase and the glucoamylase having granular starch hydrolyzing activityare active during saccharification; b) the starch substrate is used ingranular form and the starch is hydrolyzed; c) a single pH is used forsolubilization and saccharification of the granular starch; d) thesaccharification time period is shorter using granular starch comparedto the current saccharification time using liquefied starch substrate;e) a high glucose syrup with reduced higher sugar content is obtainedcompared to glucose syrup obtained from liquefied starch substrate; f)glucose loss to maltulose formation is reduced; g) milliard reactionsare eliminated or minimized; h) the risk of iodine positive starchpolymer formation (Blue-Sac), after saccharification due to retrogradedstarch formation from jet cooking is lower; i) calcium addition to thestarch slurry is eliminated; and j) filtration is improved because thehydrolyzed starch will not plug the filtration system. The methods andcompositions encompassed by the invention offer a more economical andefficient means to produce glucose feed for industrial and specialtychemicals.

In one aspect of the invention a filamentous fungal strain transformedwith a heterologous polynucleotide encoding a granular starchhydrolyzing enzyme having glucoamylase activity (GSHE) is provided. Apreferred filamentous fungal strain is a Trichoderma strain and morespecifically a T. reesei strain which expresses and secretes GSHE intothe culture medium.

In some embodiments of this aspect, the invention pertains to a methodof producing a GSHE in a filamentous host cell which comprisestransforming the filamentous fungal host cell with a DNA constructcomprising a promoter having transcriptional activity in the filamentousfungal host cell operably linked to a heterologous polynucleotideencoding a GSHE, cultivating the transformed filamentous fungal hostcell in a suitable culture medium to allow expression of the GSHE andproducing the GSHE. In other embodiments, the heterologouspolynucleotide encoding the GSHE is derived from a strain of Humicolagrisea or a strain of Aspergillus awamori. In other embodiments, theGSHE has at least 90% sequence identity to SEQ ID NO: 3 or at least 90%identity to SEQ ID NO: 6. In further embodiments, the GSHE produced bythe recombinant host cell is recovered.

In a second aspect, the invention includes a fermentation broth producedfrom a culture of recombinant Trichoderma reesei, wherein the T. reeseicomprises a heterologous polynucleotide encoding a GSHE having at least90% sequence identity to SEQ ID NO: 3 or at least 90% sequence identityto SEQ ID NO: 6.

In a third aspect, the invention pertains to a one-step process forproducing a glucose syrup from a granular starch substrate, the processcomprising (a) contacting a slurry of a granular starch substrate havinga dry solid content (ds) of 10-55% simultaneously with an alpha amylaseand a granular starch hydrolyzing enzyme having glucoamylase activity(GSHE), at a temperature equal to or below the gelatinizationtemperature of the starch substrate, and (b) allowing the alpha amylaseand the GSHE to act for a period of time sufficient to hydrolyze thegranular starch to obtain a glucose syrup. In one embodiment, at least95% of the granular starch is hydrolyzed. In a second embodiment, theyield of the glucose syrup is at least 90% by weight. In a thirdembodiment, the dry solid content of the granular starch substrate isbetween about 15 to 40%. In a fourth embodiment, the period of time tohydrolyze the granular starch is in the range of about 5 hours to 100hours. In a fifth embodiment, the alpha amylase is an enzyme having EC3.2.1.1. In a sixth embodiment, the alpha amylase is derived from aBacillus and particularly a strain of B. stearothermophilus. In furtherembodiments, the alpha amylase is derived from a recombinant Bacillusstrain. In a seventh embodiment, the GSHE is a glucoamylase derived froma Humicola grisea var. thermoidea strain or an Aspergillus awamori var.kawachi strain. In an eighth embodiment, the GSHE is a glucoamylasederived from a recombinant Trichoderma strain, and particularly a T.reesei strain which expresses a heterologous gene encoding a Humicolagrisea GSHE or an Aspergillus awamori var. kawachi GSHE. In a ninthembodiment, the process further comprises separating the glucose syrup,particularly by filtration. In a tenth embodiment, the glucose from theglucose syrup is further converted to fructose. In an eleventhembodiment, the temperature of the one-step process is conducted atabout 50 to about 70° C. In a twelfth embodiment, the pH of the processis conducted at pH 4.5 to 6.5.

In a fourth aspect, the invention relates to a one-step process forproducing a glucose syrup from a granular cornstarch substrate, theprocess comprising (a) contacting a slurry of a granular starchsubstrate having a dry solid content (ds) of 25-45% simultaneously withan alpha amylase derived from a Bacillus and a glucoamylase havinggranular starch hydrolyzing activity which is derived from a fungalsource, at a temperature of about 55 to 65° C. and a pH of about 5.0 to6.0 and allowing the alpha amylase and the glucoamylase having granularstarch hydrolyzing activity to act for a period of time sufficient tohydrolyze the granular starch to obtain a glucose syrup. In oneembodiment, at least 80% of the granular starch is hydrolyzed and theyield of glucose syrup is at least 90% by weight. In a secondembodiment, the glucoamylase is a GSHE derived from a recombinantTrichoderma reesei which expresses a heterologous polynucleotideencoding a Humicola grisea GSHE or an Aspergillus awamori var. kawachiGSHE.

In a fifth aspect, the invention relates to a method a hydrolyzinggranular starch comprising contacting a slurry of granular starch havinga dry solid content of 20-55% simultaneously with an alpha amylase and aglucoamylase having granular starch hydrolyzing activity obtained from aTrichoderma strain comprising a heterologous polynucleotide encoding aGSHE derived from Humicola grisea and allowing the alpha amylase andglucoamylase to act for a period of time sufficient to hydrolyze thegranular starch. In one embodiment, at least 90% of the granular starchis hydrolyzed. In a second embodiment, the granular starch is cornstarchor wheat starch. In a third embodiment, the GSHE is provided to theslurry at a concentration of between about 0.5 to 1.0 GSHE units ofHumicola GA/g starch; the alpha amylase is provided to the slurry at aconcentration of between about 0.1 to 0.5 kg/MT of starch, the pH of theslurry is adjusted to about pH 4.5 to 6.0; and the temperature of theslurry is adjusted to about 55 to 65° C.

In a sixth aspect, the invention relates to a method for producing aglucose syrup comprising contacting a granular starch substratesimultaneously with an alpha amylase and a granular starch hydrolyzingenzyme (GSHE), wherein the GSHE is secreted from a filamentous fungalstrain, said fungal strain comprising, a heterologous polynucleotideencoding a GSHE derived from a Humicola strain and having the amino acidsequence of at least 90% identity to SEQ ID NO: 3 to obtain a glucosesyrup. In one embodiment, the glucose is further converted to a desiredend product.

In a seventh aspect, the invention relates to an enzymatic compositioncomprising an alpha amylase and a glucoamylase having granular starchhydrolyzing activity. In one embodiment the alpha amylase is derivedfrom a Bacillus sp. and the GSHE is derived from a Humicola grisea GSHE.In a second embodiment, the GSHE is derived from a Trichoderma straingenetically engineered to comprise a polynucleotide encoding a Humicolagrisea GSHE. In a third embodiment, the pH of the composition is betweenpH 4.5 and 6.5. In a fourth embodiment, the alpha amylase is derivedfrom a B. stearothermophilus strain. In further embodiment, the ratio ofalpha amylase to GSHE in the enzyme composition is 15:1 to 1:15.

In an eighth aspect, the invention relates to a process for theproduction of a high fructose starch based syrup comprising convertingthe glucose syrup obtained by a method encompassed by the invention intoa fructose based syrup.

In a ninth aspect, the invention relates to a method of producing an endproduct wherein the glucose syrup obtained by a method encompassed bythe invention is subjected to fermentation. In some embodiments of thisaspect, the end product is an alcohol, and preferably ethanol. Infurther embodiments of this aspect, the fermentation is carried outsimultaneously with the contacting step and in other embodiments thefermentation is carried out separately and sequentially to thecontacting step. In yet further embodiments, the fermentation product isseparated from the fermentation broth.

In a tenth aspect, the invention relates to a method for producingresidual starch by separating the glucose syrup produced according tothe method of the invention and retaining the composition comprisingresidual starch. In one embodiment, the residual starch is used for theproduction of end products. In a second embodiment, the residual starchis recycled and simultaneously contacted with a GSHE and an alphaamylase at a temperature below the gelatinization temperature of thegranular starch.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the genomic DNA sequence coding for the native H. griseavar. thermoidea granular starch hydrolyzing enzyme having glucoamylaseactivity (GSHE) (SEQ ID NO: 1). The putative introns are in bold andunderlined.

FIG. 2A provides the signal sequence and mature amino acid sequence forH. grisea var. thermoidea GSHE (SEQ ID NO: 2). The putative signalsequence is in bold and underlined.

FIG. 2B provides the mature amino acid sequence for H. grisea var.thermoidea GSHE (SEQ ID NO: 3).

FIG. 3 provides an illustration of pTrex3g_N13 plasmid, which was usedfor expression of the nucleic acid encoding the Humicola grisea GSHE andwhich contains the Xba1 sites flanking the fungal expression vector,wherein

-   -   a) cbhl promoter is the Trichoderma reesei cellobiohydrolase        promoter,    -   b) H. grisea gla1 is the polynucleotide encoding the Humicola        grisea GSHE of SEQ ID NO:3,    -   c) cbhl terminator is the Trichoderma reesei cellobiohydrolase        terminator,    -   and d) amdS is an Aspergillus nidulans acetamidase marker gene.

FIG. 4 provides the nucleotide sequence (SEQ ID NO:11) (10738 bp) of thepTrex3g_N13 plasmid of FIG. 3.

FIG. 5 provides an SDS-PAGE gel indicating the expression of H. griseavar. thermoidea GSHE in a representative fermentation run forTrichoderma reesei clones as described in Example 1. Lane 1 representsthe commercial molecular weight marker, SeeBlue (Invitrogen); lane 2 isblank; lane 3 depicts rGSHE expression at 48 hours; lane 4 depicts rGSHEexpression at 56 hours; and lane 5 depicts rGSHE expression at 64 hours.

FIG. 6 provides the genomic DNA sequence coding for the Aspergillusawamori var. kawachi GSHE (SEQ ID NO: 4). The putative introns are inbold and underlined.

FIG. 7A provides the signal sequence and mature amino acid sequence forA. awamori var. kawachi GSHE (SEQ ID NO: 5). The signal sequence is inbold and underlined.

FIG. 7B provides the mature amino acid sequence for Aspergillus awamorivar. kawachi GSHE (SEQ ID NO: 6).

FIGS. 8A and 8B illustrate the pH stability as % residual activity forthe native Humicola grisea var. thermoidea GSHE (nGSHE) and theexpressed H. grisea var. thermoidea GSHE (RGSHE) in the T. reesei host(SEQ ID NO: 3), as described in Example 1.

FIG. 9 illustrates the hydrolysis of corn starch measured as mgglucose/mg protein over time for native Humicola grisea var. thermoideaGSHE and the expressed H. grisea var. thermoidea GSHE in the recombinantTrichoderma reesei host as described in Example 1.

FIG. 10 provides an SDS-PAGE gel indicating the expression ofAspergillus awamori var. kawachi GSHE in a representative fermentationrun for Trichoderma reesei clones as described in Example 2. Lane 1represents the commercial molecular weight marker, SeeBlue (Invitrogen);lane 2 depicts rGSHE expression at 162 hours, and lane 3 is a control,which depicts the untransformed Trichoderma reesei host at 162 hours.

FIG. 11 is a general diagram illustrating an embodiment of the inventiveprocess for low energy glucose production from granular starchsubstrates.

FIG. 12 illustrates a scanning electron micrograph of a typical cornstarch granule before exposure to a process of the invention (a) andscanning electron micrographs (b-d) of residual starch after exposure tothe process encompassed by the invention.

DETAILED DESCRIPTION OF THE INVENTION

In some aspects, the present invention relies on routine techniques andmethods used in the field of genetic engineering and molecular biology.The following resources include descriptions of general methodologyuseful in accordance with the invention: Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL (2nd Ed., 1989); Kreigler, GENE TRANSFERAND EXPRESSION; A LABORATORY MANUAL (1990) and Ausubel et al., Eds.CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (1994).

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton, et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley andSons, New York (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARYOF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods and materials are described.

The invention will now be described in detail by way of reference onlyusing the following definitions and examples. All patents andpublications, including all sequences disclosed within such patents andpublications, referred to herein are expressly incorporated byreference.

Numeric ranges are inclusive of the numbers defining the range.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention, which can be had by reference to thespecification as a whole.

A. DEFINITIONS

As used herein the term “starch” refers to any material comprised of thecomplex polysaccharide carbohydrates of plants, comprised of amyloseand/or amylopectin with the formula (C₆H₁₀O₅)_(x), wherein X can be anynumber. In particular, the term refers to any plant-based materialincluding but not limited to grains, grasses, tubers and roots and morespecifically the plants wheat, barely, corn, rye, rice, sorghum,legumes, cassaya, millet, potato, sweet potato, and tapioca.

The term “granular starch” refers to uncooked (raw) starch, which hasnot been subject to gelatinization.

The term “starch gelatinization” means solubilization of a starchmolecule to form a viscous suspension.

The term “gelatinization temperature” refers to the lowest temperatureat which gelatinization of a starch substrate begins. The exacttemperature depends upon the specific starch substrate and further maydepend on the particular variety of plant species from which the starchis obtained and the growth conditions.

The term “DE” or “dextrose equivalent” is an industry standard formeasuring the concentration of total reducing sugars, calculated asD-glucose on a dry weight basis. Unhydrolyzed granular starch has a DEthat is essentially 0 and D-glucose has a DE of 100.

The term “glucose syrup” refers to an aqueous composition containingglucose solids. In one embodiment, glucose syrup will include at least90% D-glucose and in another embodiment glucose syrup will include atleast 95% D-glucose. In some embodiments, the terms “glucose”, “glucosesyrup” and “dextrose” are used interchangeably.

The term “total sugar content” refers to the total sugar content presentin a starch composition.

The term “dry solids content (ds)” refers to the total solids of aslurry (in %) on a dry weight basis.

“Brix” refers to a well known hydrometer scale for measuring the sugarcontent of a solution at a given temperature. The Brix scale measuresthe number of grams of sucrose present per 100 grams of aqueous sugarsolution (the total solubilized solid content). Brix measurements arefrequently made by use of a hydrometer or refractometer.

The term “starch-liquefying enzyme” refers to an enzyme that effects thefluidization of granular starch. Exemplary starch liquefying enzymesinclude alpha amylases (E.C. 3.2.1.1).

The term “amylases” refer to enzymes that catalyze the hydrolysis ofstarches.

The term “alpha-amylase (E.C. class 3.2.1.1)” refers to enzymes thatcatalyze the hydrolysis of alpha-1,4-glucosidic linkages. These enzymeshave also been described as those effecting the exo or endohydrolysis of1,4-α-D-glucosidic linkages in polysaccharides containing 1,4-α-linkedD-glucose units. Another term used to describe these enzymes isglycogenase. Exemplary enzymes include alpha-1,4-glucan 4-glucanohydraseglucanohydrolase.

The terms “saccharification enzyme” and “glucoamylase” usedinterchangeability herein refer to the amyloglucosidase class of enzymes(EC.3.2.1.3, glucoamylase, alpha-1,4-D-glucan glucohydrolase). These areexo-acting enzymes, which release glucosyl residues from thenon-reducing ends of amylose and amylopectin molecules. The enzymes alsohydrolyze alpha-1,6 and alpha-1,3 linkages although at much slower ratesthan alpha-1,4 linkages.

The term “granular starch hydrolyzing enzyme (GSHE)” or “an enzymehaving granular starch hydrolyzing activity” as used herein specificallyrefers to an enzyme having glucoamylase activity and having the abilityto hydrolyze starch in granular form. Preferred GSHEs are those derivedfrom filamentous fungi wherein the GSHE is endogenous or exogenous tothe filamentous fungal cell. One preferred GSHE is the native GSHEderived from Humicola grisea var. thermoidea. Another preferred GSHE isderived from Aspergillus awamori var. kawachi. A particularly preferredGSHE is a recombinant GSHE, that is a GSHE expressed in a host strainthat has been genetically engineered to include a heterologouspolynucleotide encoding the GSHE. In some preferred embodiments, theGSHE is expressed as an extracellular enzyme.

The term “hydrolysis of starch” refers to the cleavage of glucosidicbonds with the addition of water molecules.

The term “degree of polymerization (DP)” refers to the number (n) ofanhydroglucopyranose units in a given saccharide. Examples of DP1 arethe monosaccharides, such as glucose and fructose. Examples of DP2 arethe disaccharides, such as maltose and sucrose. A DP4⁺ (>DP3) denotespolymers with a degree of polymerization of greater than 3.

The term “contacting” refers to the placing of the respective enzymes insufficiently close proximity to the respective substrate to enable theenzymes to convert the substrate to the end product. Those skilled inthe art will recognize that mixing solutions of the enzyme with therespective substrates can effect contacting.

The term “enzymatic conversion” in general refers to the modification ofa substrate by enzyme action. The term as used herein also refers to themodification of a granular starch substrate by the action of an enzyme.In a preferred embodiment, the enzymatic conversion of a granular starchsubstrate will result in a glucose syrup.

The term “slurry” refers to an aqueous mixture containing insolublestarch granules.

The term “glycosylation” refers to the post-transcriptional modificationof a protein by the addition of carbohydrate moieties, wherein thecarbohydrate is either N-linked or O-linked resulting in a glucoprotein.An N-linked carbohydrate moiety of a glycoprotein is attached by aglycosidic bond to the β-amide nitrogen of an asparagine residue. AnO-linked carbohydrate is attached by a glycosidic bond to a proteinthrough the hydroxy group of a serine or a threonine residue.

The term “recombinant” when used with reference e.g. to a cell, nucleicacid, protein or vector, indicates that the cell, nucleic acid, proteinor vector, has been modified by the introduction of a heterologousnucleic acid or protein or the alteration of a native nucleic acid orprotein, or that the cell is derived from a cell so modified. Thus, forexample, recombinant cells express genes that are not found within thenative (non-recombinant) form of the cell or express native genes thatare otherwise abnormally expressed, under expressed or not expressed atall.

The terms “recombinant GSHE”, “recombinantly expressed GSHE” and“recombinantly produced GSHE” refer to a mature GSHE protein sequencethat is produced in a host cell from a heterologous polynucleotide. Thesymbol “r” may be used to denote recombinant. The protein sequence of arGSHE excludes a signal sequence. In one embodiment Humicola grisea var.thermoidea GSHE expressed in a strain of Trichoderma reesei is denotedby “rH-GSHE”.

The terms “native GSHE” and “nGSHE” mean a GSHE, which was derived froma microbial host organism other than the fungal host for whichrecombinant GSHE expression is desired. Preferred native GSHEs arederived from a Humicola grisea strain or a Aspergillus awamori strain.

The terms “protein” and “polypeptide” are used interchangeably herein.The conventional one-letter or three-letter code for amino acid residuesis used herein.

A “signal sequence” means a sequence of amino acids bound to theN-terminal portion of a protein, which facilitates the secretion of themature form of the protein outside the cell. The definition of a signalsequence is a functional one. The mature form of the extracellularprotein lacks the signal sequence which is cleaved off during thesecretion process.

A “gene” refers to a DNA segment that is involved in producing apolypeptide and includes regions preceding and following the codingregions as well as intervening sequences (introns) between individualcoding segments (exons).

The term “nucleic acid” encompasses DNA, RNA, single stranded or doublestranded and chemical modifications thereof. The terms “nucleic acid”and “polynucleotide” may be used interchangeably herein. Because thegenetic code is degenerate, more than one codon may be used to encode aparticular amino acid, and the present invention encompassespolynucleotides, which encode a particular amino acid sequence.

A “vector” refers to a polynucleotide sequence designed to introducenucleic acids into one or more cell types. Vectors include cloningvectors, expression vectors, shuttle vectors, plasmids, phage particles,cassettes and the like.

An “expression vector” as used herein means a DNA construct comprising aDNA sequence which is operably linked to a suitable control sequencecapable of effecting expression of the DNA in a suitable host. Suchcontrol sequences may include a promoter to effect transcription, anoptional operator sequence to control transcription, a sequence encodingsuitable ribosome binding sites on the mRNA, enhancers and sequenceswhich control termination of transcription and translation.

A “promoter” is a regulatory sequence that is involved in binding RNApolymerase to initiate transcription of a gene. The promoter may be aninducible promoter or a constitutive promoter. A preferred promoter usedin the invention is Trichoderma reesei cbh1, which is an induciblepromoter.

“Under transcriptional control” is a term well understood in the artthat indicates that transcription of a polynucleotide sequence, usuallya DNA sequence, depends on its being operably linked to an element whichcontributes to the initiation of, or promotes transcription.

“Under translational control” is a term well understood in the art thatindicates a regulatory process that occurs after mRNA has been formed,

As used herein when describing proteins and genes that encode them, theterm for the gene is not capitalized and is italicized, e.g. the genethat encodes the Humicola grisea GSHE may be denoted as gla1. The termfor the protein is generally not italicized and the first letter iscapitalized, e.g. the protein encoded by the gla1 gene may be denoted asGla1.

The term “operably linked” refers to juxtaposition wherein the elementsare in an arrangement allowing then to be functionally related. Forexample, a promoter is operably linked to a coding sequence if itcontrols the transcription of the sequence.

The term “selective marker” refers to a gene capable of expression in ahost that allows for ease of selection of those hosts containing anintroduced nucleic acid or vector. Examples of selectable markersinclude but are not limited to antimicrobials (e.g. hygromycin,bleomycin, or chloramphenicol) or genes that confer a metabolicadvantage, such as a nutritional advantage on the host cell.

The term “derived” encompasses the terms “originated from”, “obtained orobtainable from”, and “isolated from”.

“Host strain” or “host cell” means a suitable host for an expressionvector or DNA construct comprising a polynucleotide encoding a GSHEaccording to the invention. Specifically, host strains are filamentousfungal cells. In a preferred embodiment of the invention, “host cell”means both the cells and protoplasts created from the cells of afilamentous fungal strain and particularly a Trichoderma sp. or anAspergillus sp.

The term “filamentous fungi” refers to all filamentous forms of thesubdivision Eumycotina (See, Alexopoulos, C. J. (1962), INTRODUCTORYMYCOLOGY, New York: Wiley). These fungi are characterized by avegetative mycelium with a cell wall composed of chitin, cellulose, andother complex polysaccharides. The filamentous fungi of the presentinvention are morphologically, physiologically, and genetically distinctfrom yeasts. Vegetative growth by filamentous fungi is by hyphalelongation and carbon catabolism is obligatory aerobic. In the presentinvention, the filamentous fungal parent cell may be a cell of a speciesof, but not limited to, Trichoderma, e.g., Trichoderma reesei(previously classified as T. longibrachiatum and currently also known asHypocrea jecorina), Trichoderma viride, Trichoderma koningii,Trichoderma harzianum; Penicillium sp.; Humicola sp., including Humicolainsolens and Humicola grisea; Chrysosporium sp., including C.lucknowense; Gliocladium sp.; Aspergillus sp., including A. oryzae, A.nidulans, A. niger, and A. awamori; Fusarium sp., Neurospora sp.,Hypocrea sp., and Emericella sp. Reference is also made to Innis et al.,(1985) Sci. 228:21-26.

As used herein, the term “Trichoderma” or “Trichoderma sp.” refer to anyfungal strain, which had previously been classified as Trichoderma or iscurrently classified as Trichoderma.

The term “culturing” refers to growing a population of microbial cellsunder suitable conditions in a liquid or solid medium. In oneembodiment, culturing refers to fermentative bioconversion of a granularstarch substrate to glucose syrup or other desired end products(typically in a vessel or reactor).

The term “heterologous” or “exogenous” with reference to apolynucleotide or protein refers to a polynucleotide or protein thatdoes not naturally occur in a host cell. In some embodiments, theprotein is a commercially important industrial protein. It is intendedthat the term encompass proteins that are encoded by naturally occurringgenes, mutated genes and/or synthetic genes. The term “homologous” or“endogenous” with reference to a polynucleotide or protein refers to apolynucleotide or protein that occurs naturally in the host cell.

The terms “recovered”, “isolated”, and “separated” as used herein referto a molecule, protein, cell, nucleic acid, amino acid, or carbohydratethat is removed from at least one component with which it is naturallyassociated.

As used herein, the terms “transformed”, “stably transformed” or“transgenic” with reference to a cell means the cell has a non-native(heterologous) nucleic acid sequence integrated into its genome or as anepisomal plasmid that is maintained through multiple generations.

As used herein, the term “expression” refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or “transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell where the nucleicacid sequence may be incorporated into the genome of the cell (forexample, chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed (for example,transfected mRNA).

As used herein the term “specific activity” means an enzyme unit definedas the number of moles of substrate converted to product by an enzymepreparation per unit time under specific conditions. Specific activityis expressed as units (U)/mg of protein.

As used herein “enzyme activity” refers to the action of an enzyme onits substrate.

As used herein the term “enzyme unit” refers to the amount of enzymethat converts 1 mg of substrate per minute to the substrate product atoptimum assay conditions. For example, in one embodiment, the termgranular starch hydrolyzing enzyme unit (GSHE U) is defined as being theamount of GSHE required to produce 1 mg of glucose per minute fromgranular starch under assay conditions of, for example 50° C. at pH 4.5.For example, in one embodiment, the term alpha amylase enzyme unit (AU)is defined as the amount of alpha amylase which hydrolyzes 1 micromoleof starch substrate in 1 min under standard assay conditions of pH 5.2and 40° C.

The terms “end product” or “desired end-product” refer to anycarbon-source derived molecule product which is enzymatically convertedfrom the granular starch substrate. Preferably, the end product isglucose or a glucose syrup. Glucose may then be used as a precursor forother desired end-products.

The term “residual starch” as used herein refers to the by-product orremaining components of the inventive granular starch hydrolysis processwhen the composition comprising the glucose syrup or other end productsis separated. The residual starch includes remaining insoluble starch,left in the composition after the separation.

A “residual starch recycling step” refers to the recycling of residualstarch into a vessel or reactor, which includes a GSHE and an alphaamylase.

The term “yield” refers to the amount of end-product or desiredend-products produced using the methods of the present invention. Insome preferred embodiments, the yield is greater than that producedusing methods known in the art. In some embodiments, the term refers tothe volume of the end product and in other embodiment the term refers tothe concentration of the end product.

As used herein “ethanologenic microorganism” refers to a microorganismwith the ability to convert a sugar or oligosaccharide to ethanol. Anethanologenic microorganism is ethanolgenic by virtue of their abilityto express one or more enzymes that individually or together convertsugar to ethanol.

In the present context, the term “substantially pure polypeptide” meansa polypeptide preparation which contains at the most 10% by weight ofother polypeptide material with which it is natively associated (lowerpercentages of other polypeptide material are preferred, e.g. at themost 8% by weight, at the most 6% by weight, at the most 5% by weight,at the most 4% at the most 3% by weight, at the most 2% by weight, atthe most 1% by weight, and at the most ½% by weight). Thus, it ispreferred that the substantially pure polypeptide is at least 92% pure,i.e. that the polypeptide constitutes at least 92% by weight of thetotal polypeptide material present in the preparation, and higherpercentages are preferred such as at least 94% pure, at least 95% pure,at least 96% pure, at least 96% pure, at least 97% pure, at least 98%pure, at least 99%, and at the most 99.5% pure. The polypeptidesdisclosed herein are preferably in a substantially pure form. Inparticular, it is preferred that the polypeptides disclosed herein arein “essentially pure form”, i.e. that the polypeptide preparation isessentially free of other polypeptide material with which it is nativelyassociated. This can be accomplished, for example, by preparing thepolypeptide by means of well-known recombinant methods.

“ATCC” refers to American Type Culture Collection located at Manassas,Va. 20108 (ATCC, www/atcc.org).

“NRRL” refers to the Agricultural Research Service Culture Collection,National Center for Agricultural Utilization Research (and previouslyknown as USDA Northern Regional Research Laboratory), Peoria, Ill.

“A”, “an” and “the” include plural references unless the context clearlydictates otherwise.

As used herein the term “comprising” and its cognates are used in theirinclusive sense; that is, equivalent to the term “including” and itscorresponding cognates.

B. PREFERRED EMBODIMENTS Starch Substrates

A granular starch substrate to be processed in the methods of theinvention may be obtained from any plant part including stems, grains,roots and tubers. Particularly preferred plant sources include corn;wheat; rye; sorghum; rice; millet; barley; cassava; legumes, such asbeans and peas; potatoes; sweet potatoes; bananas; and tapioca. Thestarch may be highly refined raw starch or feedstock from starchrefinery processes. Specifically contemplated starch substrates arecornstarch and wheat starch. Those of general skill in the art are wellaware of available methods which may be used to prepare granular starchsubstrates for use in the methods encompassed by the invention. Some ofthese available methods include dry milling of whole cereal grains usinghammer mills and roller mills and wet milling.

Various starches are commercially available. For example, cornstarchesare available from Cerestar, Sigma, and Katayama Chemical Industry Co.(Japan); wheat starches are available from Sigma; sweet potato starchesare available from Wako Pure Chemical Industry Co. (Japan); and potatostarch is available from Nakaari Chemical Pharmaceutical Co. (Japan).

While not meant to limit the invention in any manner, Table 1 belowprovides a general guide to the level of starch found in some commoncereal grains. As one of ordinary skill in the art is well aware thelevel of starch in a grain may vary depending on such factors asgenotype and environment.

TABLE 1 Starch Content of Various Grains Raw Material Starch % Corn60-68 Wheat 60-65 Oats 50-53 Barley 55-65 Milo 60-65 Potato 10-25Cassava 25-30 Rye 60-65 Rice (polished) 70-72 Sorghum (millet) 75-80 TheAlcohol Textbook, 3^(rd) Ed. K. Jacques et al., Eds. 1999, NottinghamUniversity Press, pg. 11.

In some embodiments of the methods encompassed by the invention, thegranular starch substrate is slurried (generally with water) and theslurry comprises i) about 10 to about 55% dry solids content, ii) about20 to about 50% dry solids content; iii) about 25 to about 45% drysolids content; iv) about 30 to about 45% dry solids content; v) about30 to about 40% dry solids content; and vi) about 30 to 35% dry solidscontent.

Alpha Amylases—

In some of the embodiments encompassed by the invention, the alphaamylase is a microbial enzyme having an E.C. number, E.C. 3.2.1.1-3 andin particular E.C. 3.2.1.1. In some embodiments, the alpha amylase is athermostable bacterial alpha amylase. Suitable alpha amylases may benaturally occurring as well as recombinant and mutant alpha amylases. Inparticularly preferred embodiments, the alpha amylase is derived from aBacillus species. Preferred Bacillus species include B. subtilis, B.stearothermophilus, B. lentus, B. licheniformis, B. coagulans, and B.amyloliquefaciens (U.S. Pat. No. 5,763,385; U.S. Pat. No. 5,824,532;U.S. Pat. No. 5,958,739; U.S. Pat. No. 6,008,026 and U.S. Pat. No.6,361,809). Particularly preferred alpha amylases are derived fromBacillus strains B. stearothermophilus, B. amyloliquefaciens and B.licheniformis. Also reference is made to strains having ATCC 39709; ATCC11945; ATCC 6598; ATCC 6634; ATCC 8480; ATCC 9945A and NCIB 8059.

Commercially available alpha amylases contemplated for use in thecompositions and methods of the invention include; SPEZYME AA; SPEZYMEFRED; GZYME G997 (Genencor International Inc.) and TERMAMYL 120-L, LC,SC and SUPRA (Novozyme Biotech).

As understood by those in the art, the quantity of alpha amylase used inthe compositions and methods of the present invention will depend on theenzymatic activity of the alpha amylase. In general, an amount of about0.01 to 5.0 kg of the alpha amylase is added to a metric ton (MT) of theraw material (granular starch substrate). This amount is approximatelyequivalent to 0.06 AU/g ds to 30 AU/g ds with a GZYME 997. In someembodiments, the alpha amylase is added in an amount about 0.05 to 5.0kg; about 0.05 to 2.5 kg; about 0.1 to 2.5 kg; about 0.1 to 2.0 kg;about 0.1 to 1.5 kg; about 0.1 to 1.0 kg; about 0.5 to 5.0 kg and about0.5 to 2.0 kg per metric ton. These values are approximately equal to0.3 to 30 AU/g ds; 0.3 to 15 AU/g ds; 0.6 to 15 AU/g ds; 0.6 to 12 AU/gds; 0.6 to 9 AU/g ds; 0.6 to 6 AU/g ds; 3 to 30 AU/g ds and also 3 to 12AU/g ds with a GZYME 997. In further embodiments, other quantities areutilized, for example, generally an amount of between about 0.01 to 1.0kg of GZYME 997 or SPEZYME FRED (Genencor International Inc.) is addedto a metric ton of starch. In other embodiments, the enzyme is added inan amount between about 0.05 to 1.0 kg; between about 0.1 to 0.6 kg;between about 0.2 to 0.6 kg and between about 0.4 to 0.6 kg of GZYME 997or SPEZYME FRED per metric ton of starch.

Granular Starch Hydrolyzing Enzymes Having Glucoamylase Activity—

Glucoamylases (E.C. 3.2.1.3) are enzymes that remove successive glucoseunits from the non-reducing ends of starch. The enzyme can hydrolyzeboth linear and branched glucosidic linkages of starch, amylose andamylopectin. While glucoamylase may be derived from bacteria, plants andfungi, preferred glucoamylases encompassed by the present are derivedfrom fungal strains.

Glucoamylases secreted from fungi of the genera Aspergillus, Rhizopus,Humicola and Mucor have been derived from fungal strains, includingAspergillus niger, Aspergillus awamori, Rhizopus niveus, Rhizopusoryzae, Mucor miehe, Humicola grisea, Aspergillus shirousami andHumicola (Thermomyces) laniginosa (See, Boel et al. (1984) EMBO J.3:1097-1102; WO 92/00381; WO 00/04136; Chen et al., (1996) Prot. Eng.9:499-505; Taylor et al., (1978) Carbohydrate Res. 61:301-308 and Jensenet al, (1988) Can. J. Microbiol. 34:218-223).

Enzymes having glucoamylase activity used commercially are produced forexamples, from Aspergillus niger (trade name OPTIDEX L-400 and G ZYMEG990 4X from Genencor International Inc.) or Rhizopus species (tradename CU.CONC. from Shin Nihon Chemicals, Japan and trade name GLUCZYMEfrom Amano Pharmaceuticals, Japan).

A particular group of enzymes having glucoamylase activity are granularstarch hydrolyzing enzyme(s) GSHE (See, Tosi et al, (1993) Can. J.Microbiol. 39:846-855). GSHEs not only have glucoamylase activity, butalso are able to hydrolyze granular (raw) starch. GSHEs have beenrecovered from fungal cells such as Humicola sp., Aspergillus sp. andRhizopus sp. A Rhizopus oryzae GSHE has been described in Ashikari etal., (1986) Agric. Biol. Chem. 50:957-964 and U.S. Pat. No. 4,863,864. AHumicola grisea GSHE has been described in Allison et al., (1992) Curr.Genet. 21:225-229 and European Patent No. 171218. The gene encoding thisenzyme is also known in the art as gla1. An Aspergillus awamori var.kawachi GSHE has been described by Hayashida et al., (1989) Agric. Biol.Chem. 53:923-929. An Aspergillus shirousami GSHE has been described byShibuya et al., (1990) Agric. Biol. Chem. 54:1905-1914.

In one embodiment a GSHE may be derived from a strain of Humicolagrisea, particularly a strain of Humicola grisea var. thermoidea (see,U.S. Pat. No. 4,618,579).

In some preferred embodiments, the GSHE is recovered from fungiincluding ATCC 16453, NRRL 15219, NRRL 15220, NRRL 15221, NRRL 15222,NRRL 15223, NRRL 15224 and NRRL 15225 as well as genetically alteredstrains thereof. (EP 0 171218).

In one embodiment, a GSHE may be derived from a strain of Aspergillusawamori, particularly a strain of A. awamori var. kawachi (See,Hayashida et al., (1989) Agric. Biol. Chem. 53:923-929).

In another embodiment, a GSHE may exhibit a maximum pH activity within apH range of 4 to 7.5 and within a pH range of 5.0 to 7.5 and a maximumactivity in the temperature range of 50 to 60° C.

In one embodiment, the GSHE has at least 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 93%, 95%, 97%, 98% and 99% sequence identity with theamino acid sequence set forth in SEQ ID NO: 3. In another embodiment,the GSHE comprises an amino acid sequence having at least 80% sequenceidentity with the sequence set forth in SEQ ID NO: 3. In otherembodiments, the GSHE comprising the amino acid sequence of SEQ ID NO: 3or a GSHE having at least 80% sequence identity with the sequence of SEQID NO: 3 is encoded by a polynucleotide having at least 70%, 80%, 85%,90%, 93%, 95%, 97%, 98% and 99% sequence identity with SEQ ID NO: 1.

In another embodiment, the GSHE has at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 93%, 95%, 97%, 98% and 99% sequence identity withthe amino acid sequence set forth in SEQ ID NO: 6. In anotherembodiment, the GSHE comprises an amino acid sequence having at least80% sequence identity with the sequence set forth in SEQ ID NO: 6. Inother embodiments, the GSHE comprising the amino acid sequence of SEQ IDNO: 6 or a GSHE having at least 80% sequence identity with the sequenceof SEQ ID NO: 6 is encoded by a polynucleotide having at least 70%, 80%,85%, 90%, 93%, 95%, 97%, 98% and 99% sequence identity with SEQ ID NO:4.

A polynucleotide or polypeptide having a certain percent (e.g., 80%,85%, 90% or 99%) of sequence identity with another sequence means thatwhen aligned, that percent of bases or amino acid residues are the samein comparing the two sequences. This alignment and the percent homologyor identity can be determined using any suitable software program knownin the art, for example those described in Current Protocols inMolecular Biology (Ausubel et al., eds 1987 Supplement 30, section7.7.18). Preferred programs include GCG Pileup program, FASTA and BLAST.Another preferred alignment program is ALIGN Plus and TFASTA.

One skilled in the art will recognize that sequences encompassed by theinvention are also defined by the ability to hybridize under stringenthybridization conditions with the exemplified GSHE sequences (e.g. SEQID NO: 1 or SEQ ID NO: 4). A nucleic acid is hybridizable to anothernucleic acid sequence when a single stranded form of the nucleic acidcan anneal to the other nucleic acid under appropriate conditions oftemperature and solution ionic strength. Hybridization and washingconditions are well known in the art (See, e.g. Sambrook (1989) supra,particularly chapters 9 and 11). In some embodiments, stringentconditions correspond to a Tm of 65° C. and 0.1×SSC, 0.1% SDS. In afurther embodiment, a GSHE enzyme may be derived from a strain ofRhizopus. Such as the enzyme derived from the Koji strain of R. niveus(sold under the trade name “CU CONC”) or the enzyme from Rhizopus soldunder the trade name GLUCZYME.

In a preferred embodiment, the GHSE used in a method or compositionencompassed by the invention is a recombinantly expressed GSHE obtainedfrom a filamentous fungal strain, which has been genetically engineeredto express a heterologous polynucleotide that encodes a GSHE derivedfrom a source other than the host strain.

In some embodiments the filamentous fungal strain is a strain ofAspergillus sp., Trichoderma sp., Fusarium sp., or Penicillium sp.Particularly preferred fungal hosts include A. nidulans, A. awamori, A.oryzae, A. aculeatus, A. niger, A. japonicus, T. reesei, T. viride, Foxysporum, and F. solani. Aspergillus strains are disclosed in Ward etal., (1993) Appl. Microbiol. Biotechnol. 39:738-743 and Goedegebuur etal., (2002) Curr. Gene 41:89-98. In a most preferred embodiment, thehost is a Trichoderma strain and particularly a T. reesei strain.Strains of T. reesei are known and nonlimiting examples include ATCC No.13631, ATCC No. 26921, ATCC No. 56764, ATCC No. 56765, ATCC NO. 56767and NRRL 15709. In some preferred embodiments, the host strain is aderivative of RL-P37. RL-P37 is disclosed in Sheir-Neiss et al., (1984)Appl. Microbiol. Biotechnol. 20:46-53.

A host strain which expresses rGSHE may have be previously manipulatedthrough genetic engineering. In some embodiments, various genes of thefungal host have been inactivated. These genes include, for examplegenes encoding cellulolytic enzymes, such as endoglucanases (EG) andexocellobiohydrolases (CBH) (e.g., cbh1, cbh2, egl1, egl2 and egl3).U.S. Pat. No. 5,650,322 discloses derivative strains of RL-P37 havingdeletions in the cbh1 gene and the cbh2 gene.

In some embodiments, the fungal host has been genetically engineered tocomprise a polynucleotide encoding a GSHE derived from Humicola grisea.In one embodiment the rGSHE will have at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 93%, 95%, 97%, 98% and 99% sequence identity to theamino acid sequence set forth in SEQ ID NO: 3. In other embodiments, apolynucleotide encoding the GSHE of SEQ ID NO: 3 will have at least 70%,80%, 85%, 90%, 95%, 97% and 98% sequence identity with the sequence ofSEQ ID NO: 1. In a particularly preferred embodiment, the GSHE isexpressed in a Trichoderma reesei strain and the produced protein has atleast 80%, 85%, 90%, 95%, 97% and 98% sequence identity with thesequence of SEQ ID NO: 3.

In other embodiments, the fungal host has been genetically engineered toexpress a polynucleotide encoding a GSHE derived from Aspergillusawamori. In one embodiment, the rGSHE will have at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 97%, 98% and 99% sequenceidentity to the amino acid sequence set forth in SEQ ID NO. 6. In otherembodiments, a polynucleotide encoding the GSHE of SEQ ID NO: 6 willhave at least 70%, 80%, 85%, 90%, 95%, 97% and 98% sequence identitywith the sequence of SEQ ID NO: 4. In a particularly preferredembodiment, the GSHE is expressed in a Trichoderma reesei strain and theproduced protein has at least 80%, 85%, 90%, 95%, 97% and 98% sequenceidentity with the sequence of SEQ ID NO: 6.

In some embodiments, the level of glycosylation of the recombinantlyexpressed GSHE is different that the level of glycosylation of thecorresponding native GSHE (e.g., GSHE which was originally derived fromH. grisea or A. awamori has a different level of glycosylation than thelevel of glycosylation of the GSHE expressed in Trichoderma). In oneembodiment, the level of glycosylation is different even if the rGSHEhas at least 80%, 85%, 90%, 95% amino acid identity to the correspondingnative GSHE. In some embodiments, a rGSHE expressed in Trichoderma andparticularly a strain of T. reesei has a different level ofglycosylation than the level from the corresponding nGSHE. In otherembodiments, the level of glycosylation is higher, while in otherembodiments it is lower.

For example, the level of glycosylation for rGSHE may be at least 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% lessthan the level of glycosylation of the corresponding nGSHE. In otherembodiments, the level of glycosylation of an expressed rGSHE may be atleast 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%,100%, 125% 150%, 175%, 200%, 225% or 250% greater than the level of acorresponding nGSHE.

In another embodiment, the recombinantly produced GSHE producedaccording to the invention may have greater stability at lower pH levelsthan the corresponding native GSHE at optimum temperature levels. Morespecifically, a rGSHE expressed in Trichoderma, which was originallyderived from Humicola grisea var. thermoidea, has a greater stability atpH levels of 3.5 to 4.0 compared to a corresponding nGSHE at atemperature of 45-55° C. Under some conditions the stability of rGSHEand particularly H. grisea var. thermoidea SEQ ID NO: 1 expressed inTrichoderma reesei is more than double the level of stability of nGSHE.

Vectors and Fungal Transformation:

According to the invention, a DNA construct comprising a polynucleotideencoding a GSHE encompassed by the invention is constructed to transferGSHE into a host cell. Thus, a GSHE polynucleotide which can beexpressed in enzyme form may be introduced into a host cell using avector, particularly an expression vector which comprises a regulatorysequence operably linked to a GSHE coding sequence.

The vector may be any vector which when introduced into a fungal hostcell is integrated into the host cell genome and is replicated.Reference is made to the Fungal Genetics Stock Center Catalogue ofStrains (www.FGSC. net) for a list of vectors. Also, examples ofsuitable expression vectors may be found in Sambrook et al., (1989)supra, and Ausubel (1987) supra, and more specifically reference is madeto van den Hondel et al. (1991) in Bennett and Lasure Eds. MORE GENEMANIPULATIONS IN FUNGI, Academic Press pp. 396-428. Particularly usefulvectors include pFB6, pBR322, PUC18, pUC100 and pENTR/D.

In some embodiments, a nucleic acid encoding a GSHE is operably linkedto a suitable promoter, which shows transcriptional activity in thefungal host cell. The promoter may be derived from genes encodingproteins either homologous or heterologous to the host cell. Preferably,the promoter is useful in a Trichoderma host and suitable nonlimitingexamples of promoters include cbh1, cbh2, egl1, egl2. In one embodiment,the promoter is one that is native to the host cell. For example, whenT. reesei is the host, the promoter would be a native T. reeseipromoter. In a preferred embodiment, the promoter is T. reesei cbh1,which is an inducible promoter and has been deposited in GenBank underAccession No. D86235. An inducible promoter is a promoter that is activeunder environmental or developmental regulation. In another embodimentthe promoter is one that is heterologous to the fungal host cell. Otherexamples of useful promoters include promoters from A. awamori and A.niger glucoamylase genes (See, Nunberg et al., (1984) Mol. Cell. Biol.4:2306-2315 and Boel et al., (1984) EMBO J. 3:1581-1585). Also, thepromoters of the T. reesei xln1 gene and the cellobiohydrolase 1 genemay be useful (EPA 137280A1).

In some embodiments, the GSHE coding sequence is operably linked to asignal sequence. The DNA encoding the signal sequence is preferably thatwhich is naturally associated with the GSHE gene to be expressed.Preferably, the signal sequence is encoded by a Humicola grisea orAspergillus awamori gene which encodes a GSHE. More preferably thesignal sequence has at least 90%, at least 95%, at least 97%, and atleast 99% sequence identity to the signal sequence depicted in FIGS. 2Aand 6A. In additional embodiments, a signal sequence and a promotersequence comprising a DNA construct or vector to be introduced into afungal host cell are derived from the same source. For example, in someembodiments, the signal sequence is the cdh1 signal sequence which isoperably linked to a cdh1 promoter.

In some embodiments, the expression vector also includes a terminationsequence. In one embodiment, the termination sequence and the promotersequence are derived from the same source. In another embodiment, thetermination sequence is homologous to the host cell. A particularlysuitable terminator sequence is cbh1 derived from a Trichoderma strainand particularly T. reesei. Other useful fungal terminators include theterminator from A. niger or A. awamori glucoamylase gene (Nunberg et al.(1984) supra, and Boel et al., (1984) supra).

In some embodiments, an expression vector includes a selectable marker.Examples of preferred selectable markers include ones which conferantimicrobial resistance (e.g., hygromycin and phleomycin). Nutritionalselective markers also find use in the present invention including thosemarkers known in the art as amdS, argB and pyr4. Markers useful invector systems for transformation of Trichoderma are described inFinkelstein, chapter 6 in BIOTECHNOLOGY OF FILAMENTOUS FUNGI,Finkelstein et al. Eds. Butterworth-Heinemann, Boston, Mass. (1992),Chap. 6. and Kinghorn et al. (1992) APPLIED MOLECULAR GENETICS OFFILAMENTOUS FUNGI, Blackie Academic and Professional, Chapman and Hall,London. In a preferred embodiment, the selective marker is the amdSgene, which encodes the enzyme acetamidase allowing transformed cells togrow on acetamide as a nitrogen source. (See, Kelley et al., (1985) EMBOJ. 4:475-479 and Penttila et al., (1987) Gene 61:155-164.

An expression vector comprising a polynucleotide encoding a GSHE may beany vector which is capable of replicating autonomously in a givenfungal host organism or of integrating into the DNA of the host. In someembodiments, an expression vector is a plasmid. In preferredembodiments, two types of expression vectors for obtaining expression ofgenes are contemplated.

The first expression vector comprises DNA sequences in which thepromoter, GSHE coding region, and terminator all originate from the geneto be expressed. In some embodiments, gene truncation is obtained bydeleting undesired DNA sequences (e.g., coding for unwanted domains) toleave the domain to be expressed under control of its owntranscriptional and translational regulatory sequences.

The second type of expression vector is preassembled and containssequences required for high-level transcription and a selectable marker.In some embodiments, the coding region for a GSHE gene or part thereofis inserted into this general-purpose expression vector such that it isunder the transcriptional control of the expression constructs promoterand terminator sequences. In some embodiments, genes or part thereof areinserted downstream of the strong cbh1 promoter.

Methods used to ligate a vector comprising a polynucleotide encoding aGSHE, a promoter, a terminator and other sequences and to insert theminto a suitable vector are well known in the art. Linking is generallyaccomplished by ligation at convenient restriction sites. If such sitesdo not exist, the synthetic oligonucleotide linkers are used inaccordance with conventional practice. (See, Sambrook (1989) supra, andBennett and Lasure, MORE GENE MANIPULATIONS IN FUNGI, Academic Press,San Diego (1991) pp 70-76.) Additionally, vector can be constructedusing known recombination techniques (e.g. Invitrogen Life Technologies,Gateway Technology).

Where it is desired to obtain a fungal host cell having one or moreinactivated genes known methods may be used (See, U.S. Pat. No.5,246,853; U.S. Pat. No. 5,475,101 and WO 92/06209). Gene inactivationmay be accomplished by complete or partial deletion, by insertionalinactivation or by any other means which renders a gene nonfunctionalfor its intended purpose (such that the gene is prevented fromexpression of a functional protein). Any gene from a Trichoderma sp. orother filamentous fungal host, which has been cloned can be deleted, forexample cbh1, cbh2, egl1 and egl2. In some embodiments, gene deletion isaccomplished by inserting a form of the desired gene to be inactivatedinto a plasmid by known methods. The deletion plasmid is then cut at anappropriate restriction enzyme site(s), internal to the desired genecoding region and the gene coding sequence or part thereof id replacedwith a selectable marker, Flanking DNA sequences from the locus of thegene to be deleted remain on either side of the market (preferably aboutbetween 0.5 to 2.0 kb). An appropriate deletion plasmid will generallyhave unique restriction enzyme sites present therein to enable thefragment containing the deleted gene, including the flanking DNAsequences and the selectable marker gene to be removed as a singlelinear piece.

Introduction of a DNA construct or vector into a host cell includestechniques such as transformation; electroporation; nuclearmicroinjection; transduction; transfection, including lipofectionmediated and DEAE-Dextrin mediated transfection; incubation with calciumphosphate DNA precipitate; high velocity bombardment with DNA-coatedmicroprojectiles; and protoplast fusion. General transformationtechniques are taught in Ausubel et al., (1987), supra chapter 9 andSambrook (1989) supra. More specifically methods of transformation forfilamentous fungi are disclosed in Campbell et al., (1989) Curr. Genet.16:53-56. Specifically, to effect the expression of heterologous proteinin Trichoderma reference is made to U.S. Pat. No. 6,022,725; U.S. Pat.No. 6,268,328; Harkki et al. (1991); Enzyme Microb. Technol. 13:227-233;Harkki et al., (1989) Bio Technol. 7:596-603; EP 244,234; and EP215,594. Reference is also made to Nevalainen et al., “The MolecularBiology of Trichoderma and its Application to the Expression of BothHomologous and Heterologous Genes”, in MOLECULAR INDUSTRIAL MYCOLOGY,Eds. Leong and Berka, Marcel Dekker Inc., NY (1992) pp. 129-148.Reference is also made to Cao et al., (2000) Sci. 9:991-1001 fortransformation of Aspergillus strains.

Preferably genetically stable transformants may be constructed withvector systems whereby the nucleic acid encoding GSHE is stablyintegrated into a host strain chromosome. Transformants may then bepurified by known techniques.

In one nonlimiting example, stable transformants including an amdSmarker are distinguished from unstable transformants by their fastergrowth rate and the formation of circular colonies with a smooth, ratherthan ragged outline on solid culture medium containing acetamide.Additionally, in some cases a further test of stability is conducted bygrowing the transformants on solid non-selective medium (i.e. lackingacetamide), harvesting spores from this culture medium and determiningthe percentage of these spores which will subsequently germinate andgrow on selective medium containing acetamide. Alternatively, othermethods known in the art may be used to select transformants.

In one specific embodiment, the preparation of Trichoderma sp. fortransformation involves the preparation of protoplasts from fungalmycelia. (See, Campbell et al., (1989) Curr. Genet. 16:53-56). In someembodiments, the mycelia are obtained from germinated vegetative sporesand treated with an enzyme that digests the cell wall resulting inprotoplasts. The protoplasts are then protected by the presence of anosmotic stabilizer in the suspending medium. These stabilizers includesorbitol, mannitol, potassium chloride, magnesium sulfate and the like.Usually the concentration of these stabilizers varies between 0.8 M and1.2 M. It is preferable to use about a 1.2 M solution of sorbitol in thesuspension medium.

Uptake of DNA into the host Trichoderma sp. strain is dependent upon thecalcium ion concentration. Generally between about 10 mM CaCl₂ and 50 mMCaCl₂ is used in an uptake solution. Besides the need for the calciumion in the uptake solution, other items generally included are abuffering system such as TE buffer (10 Mm Tris, pH 7.4; 1 mM EDTA) or 10mM MOPS, pH 6.0 buffer (morpholinepropanesulfonic acid) and polyethyleneglycol (PEG). It is believed that the polyethylene glycol acts to fusethe cell membranes thus permitting the contents of the medium to bedelivered into the cytoplasm of the Trichoderma sp. strain and theplasmid DNA is transferred to the nucleus. This fusion frequently leavesmultiple copies of the plasmid DNA tenderly integrated into the hostchromosome.

Usually a suspension containing the Trichoderma sp. protoplasts or cellsthat have been subjected to a permeability treatment at a density of 10⁵to 10⁷/mL, preferably 2×10⁶/mL are used in transformation. A volume of100 μL of these protoplasts or cells in an appropriate solution (e.g.,1.2 M sorbitol; 50 mM CaCl₂) are mixed with the desired DNA. Generally ahigh concentration of PEG is added to the uptake solution. From 0.1 to 1volume of 25% PEG 4000 can be added to the protoplast suspension.However, it is preferable to add about 0.25 volumes to the protoplastsuspension. Additives such as dimethyl sulfoxide, heparin, spermidine,potassium chloride and the like may also be added to the uptake solutionand aid in transformation. Similar procedures are available for otherfungal host cells. See, for example, U.S. Pat. Nos. 6,022,725 and6,268,328, the contents of which are hereby incorporated by reference.

Generally, the mixture is then incubated at approximately 0° C. for aperiod of between 10 to 30 minutes. Additional PEG is then added to themixture to further enhance the uptake of the desired gene or DNAsequence. The 25% PEG 4000 is generally added in volumes of 5 to 15times the volume of the transformation mixture; however, greater andlesser volumes may be suitable. The 25% PEG 4000 is preferably about 10times the volume of the transformation mixture. After the PEG is added,the transformation mixture is then incubated either at room temperatureor on ice before the addition of a sorbitol and CaCl₂ solution. Theprotoplast suspension is then further added to molten aliquots of agrowth medium. This growth medium permits the growth of transformantsonly.

Cell Culture—

Appropriate host cells are generally cultured in a standard mediumcontaining physiological salts and nutrients, such as described inPourquie, J. et al., BIOCHEMISTRY AND GENETICS OF CELLULOSE DEGRADATION,eds. Aubert, J. P. et al., Academic Press, pp. 71-86, 1988 and Ilmen, M.et al., (1997) Appl. Environ. Microbiol. 63:1298-1306. Also reference ismade to common commercially prepared media such as Yeast Malt Extract(YM) broth, Luria Bertani (LB) broth and Sabouraud Dextrose (SD) broth.

Culture conditions are also standard, e.g., cultures are incubated atapproximately 28° C. in appropriate media in shaker cultures orfermenters until desired levels of GSHE expression are achieved.Preferred culture conditions for a given filamentous fungus may be foundin the scientific literature and/or from the source of the fungi such asthe American Type Culture Collection and Fungal Genetics Stock Center(www.FGSC.net).

After fungal growth has been established, the cells are exposed toconditions effective to cause or permit the expression of a GSHE andparticularly a GSHE as defined herein. In cases where a GSHE codingsequence is under the control of an inducible promoter, the inducingagent, e.g., a sugar, metal salt or antibiotics, is added to the mediumat a concentration effective to induce GSHE expression.

Industrial Uses of the rGSHE—Fermentations—

In some embodiments of the present invention, fungal cells expressing aheterologous GSHE are grown under batch or continuous fermentationconditions. A classical batch fermentation is a closed system, whereinthe composition of the medium is set at the beginning of thefermentation and is not subject to artificial alterations during thefermentation. Thus, at the beginning of the fermentation the medium isinoculated with the desired organism(s). In this method, fermentation ispermitted to occur without the addition of any components to the system.Typically, a batch fermentation qualifies as a “batch” with respect tothe addition of the carbon source and attempts are often made atcontrolling factors such as pH and oxygen concentration. The metaboliteand biomass compositions of the batch system change constantly up to thetime the fermentation is stopped. Within batch cultures, cells progressthrough a static lag phase to a high growth log phase and finally to astationary phase where growth rate is diminished or halted. Ifuntreated, cells in the stationary phase eventually die. In general,cells in log phase are responsible for the bulk of production of endproduct.

A variation on the standard batch system is the “fed-batch fermentation”system, which also finds use with the present invention. In thisvariation of a typical batch system, the substrate is added inincrements as the fermentation progresses. Fed-batch systems are usefulwhen catabolite repression is apt to inhibit the metabolism of the cellsand where it is desirable to have limited amounts of substrate in themedium. Measurement of the actual substrate concentration in fed-batchsystems is difficult and is therefore estimated on the basis of thechanges of measurable factors such as pH, dissolved oxygen and thepartial pressure of waste gases such as CO₂. Batch and fed-batchfermentations are common and well known in the art.

Continuous fermentation is an open system where a defined fermentationmedium is added continuously to a bioreactor and an equal amount ofconditioned medium is removed simultaneously for processing. Continuousfermentation generally maintains the cultures at a constant high densitywhere cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth and/or end productconcentration. For example, in one embodiment, a limiting nutrient suchas the carbon source or nitrogen source is maintained at a fixed rate anall other parameters are allowed to moderate. In other systems, a numberof factors affecting growth can be altered continuously while the cellconcentration, measured by media turbidity, is kept constant. Continuoussystems strive to maintain steady state growth conditions. Thus, cellloss due to medium being drawn off must be balanced against the cellgrowth rate in the fermentation. Methods of modulating nutrients andgrowth factors for continuous fermentation processes as well astechniques for maximizing the rate of product formation are well knownin the art of industrial microbiology.

Identification of GSHE Activity—

In order to evaluate the expression of a GSHE by a cell line that hasbeen transformed with a heterologous polynucleotide encoding a GSHEencompassed by the invention, assays can be carried out at the proteinlevel, the RNA level or by use of functional bioassays particular toglucoamylase activity and/or production.

In general, assays employed to analyze the expression of a GSHE include,Northern blotting, dot blotting (DNA or RNA analysis), RT-PCR (reversetranscriptase polymerase chain reaction), or in situ hybridization,using an appropriately labeled probe (based on the nucleic acid codingsequence) and conventional Southern blotting and autoradiography.

In addition, the production and/or expression of a GSHE may be measuredin a sample directly, for example, by assays directly measuring reducingsugars such as glucose in the culture media and by assays for measuringglucoamylase activity, expression and/or production. Substrates usefulfor assaying GSHE activity include granular starch substrates. Forexample, glucose concentration may be determined by any convenientmethod such as by using glucose reagent kit No 15-UV (Sigma ChemicalCo.) or an instrument such as

Technicon Autoanalyzer. Also reference is made to glucose oxidase kitsand glucose hexose kits commercially available from Instrumentation Lab.(Lexington, Mass.). Glucoamylase activity may be assayed by the3,5-dinitrosalicylic acid (DNS) method (See, Goto et al., (1994) Biosci.Biotechnol. Biochem. 58:49-54). In one nonlimiting example, a rGSHE hasthe ability to hydrolyze granular starch in a 15% starch solidssuspension in water to a solution of saccharides of at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% wt glucose, dry substancebasis.

In an embodiment of the invention, the GSHE expressed by a recombinanthost will be greater than 1 gram protein per liter (g/L) of culturemedia. Preferably in some embodiments, the host is a Trichoderma or anAspergillus host. In some embodiments, the amount of GSHE expressed by arecombinant Trichoderma host will be greater than 2 g/L of culturemedia. In other embodiments, the amount of GSHE expressed by arecombinant Trichoderma host will be greater than 5 g/L of culturemedia. Yet in other embodiments the amount of GSHE expressed by arecombinant Trichoderma host will be greater than 10 g/L of culturemedia.

The amount of expressed GSHE may in some instances be greater than 20g/L, greater than 25 g/L, greater than 30 g/L and greater than 50 g/L ofculture media.

In addition, protein expression, may be evaluated by immunologicalmethods, such as immunohistochemical staining of cells, tissue sectionsor immunoassay of tissue culture medium, e.g., by Western blot or ELISA.

Such immunoassays can be used to qualitatively and quantitativelyevaluate expression of a GSHE. The details of such methods are known tothose of skill in the art and many reagents for practicing such methodsare commercially available.

Exemplary assays include ELISA, competitive immunoassays,radioimmunoassays, Western blot, indirect immunofluorescent assays andthe like. In general, commercially available antibodies and/or kits maybe used for the quantitative immunoassay of the expression level of aGSHE.

Methods for Purifying GSHE—

In general, a GSHE (nGSHE or rGSHE) produced in cell culture is secretedinto the medium and may be purified or isolated, e.g., by removingunwanted components from the cell culture medium. In some cases, a GSHEmay be produced in a cellular form necessitating recovery from a celllysate. In such cases the enzyme is purified from the cells in which itwas produced using techniques routinely employed by those of skill inthe art. Examples include, but are not limited to, affinitychromatography (Tilbeurgh et al., (1984) FEBS Lett. 16:215);ion-exchange chromatographic methods (Goya) et al., (1991) Biores.Technol. 36:37; Fliess et al., (1983) Eur. J. Appl. Microbiol.Biotechnol. 17:314; Bhikhabhai et al., (1984) J. Appl. Biochem. 6:336;and Ellouz et al., (1987) Chromatography 396:307), includingion-exchange using materials with high resolution power (Medve et al.,(1998) J. Chromatography A 808:153; hydrophobic interactionchromatography (Tomaz and Queiroz, (1999) J. Chromatography A 865:123;two-phase partitioning (Brumbauer, et al., (1999) Bioseparation 7:287);ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; and gel filtration using, e.g., SephadexG-75. The degree of purification desired will vary depending on the useof the GSHE. In some embodiments, purification will not be necessary.

In some embodiments, the recombinantly expressed GSHE is from aTrichoderma host. In other embodiments the Trichoderma host expresses aheterologous polynucleotide, which encodes a GSHE from a Humicola griseastrain, particularly a strain of Humicola grisea var. thermoidea. Inother embodiments, the Trichoderma expresses a recombinant GSHE whereinthe heterologous polynucleotide encodes a GSHE having at least 50%sequence identity with the sequence of SEQ ID NO:3.

In some embodiments, Trichoderma host expresses a heterologouspolynucleotide, which encodes a GSHE from a Aspergillus awamori strain,particularly a strain of A. awamori var. kawachi. In other embodiments,the Trichoderma expresses a recombinant GSHE wherein the heterologouspolynucleotide encodes a GSHE having at least 50% sequence identity withthe sequence of SEQ ID NO:6.

Composition and Process Conditions—

Whether the GSHE is supplied in a cell free extract or supplied in theculture medium (fermentation broth), which includes fungal cells thatexpress and secret GSHE, the granular starch substrate, preferably inslurry form is contacted with the GSHE and alpha amylase essentiallysimultaneously (referred to herein as simultaneously) to hydrolyze thegranular starch and produce a glucose syrup. The hydrolysis of thegranular starch is a one-step process.

A GSHE may be added to a composition comprising an alpha amylase and agranular starch substrate in an amount of between about 0.01 to 10.0GSHE U/g starch dry solids of a slurry adjusted to 10-55% dry solids. Insome embodiments, the GSHE is added in an amount of between about 0.01and 5.0 GSHE U/g; about 0.01 and 2.0 GSHE U/g; about 0.01 and 1.5 GSHEU/g; about 0.05 and 1.5 GSHE U/g; about 0.1 and 5.0 GSHE U/g; about 0.1and 1.0 GSHE U/g; about 0.25 and 2.5 GSHE U/g; about 0.5 and 5.0 GSHEU/g; and about 0.5 and 1.0 GSHE U/g of such solution. Also in somepreferred embodiments, the GSHE is added in an amount of between about0.05 and 1.5 GSHE U/g of such solution, also between 0.1 and 2.0 GSHEU/g and also between about 0.1 and 1.0 GSHE U/g.

In further embodiments, a GSHE is added to a granular starch slurrycomposition essentially simultaneously with alpha amylase wherein theslurry is adjusted to 10 to about 55% ds, preferably 20-45% ds and also25-45% ds. In certain embodiments, the alpha amylase comprising thecomposition is added in a range of about 0.01 to 1.0 kg of GZYME 997 permetric ton of starch.

In one embodiment, the granular starch substrate is contacted with aGSHE wherein the GSHE is available as a cell free filtrate (such thatthe GSHE is isolated from the culture medium). In another embodiment,the granular starch substrate is contacted with a GSHE, wherein the GSHEis available in a culture medium containing the secreted GSHE and fungalcells. Preferably, the GSHE will be secreted from a Trichoderma reeseicontaining a heterologous polynucleotide encoding a polypeptide havinggranular starch hydrolyzing activity and at least 90%, at least 95% andat least 98% sequence identity with the sequence of SEQ ID NO: 3 or SEQID NO: 6.

The methods of the invention are conducted at a temperature equal to orbelow the gelatinization temperature of the granular starch of thesubstrate. In some embodiments, the method is conducted at a temperatureof at least about 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55°C., 60° C., 65° C., 70° C. and 75° C. In other embodiments, the methodis conducted at a temperature less than about 65° C. and also less thanabout 60° C. In other embodiments, the method is conducted at atemperature of between about 30° C. and 65° C.; also between about 35°C. and 65° C.; between about 40° C. and 65° C.; between about 45° C. and65° C.; and between about 50° C. and 65° C. The exact temperature usedin accordance with the method depends upon the specific starch substrateand further may depend upon the particular plant variety. In someembodiments, when corn is the granular starch substrate the temperatureis conducted at about between 55° C. and 65° and more particularlybetween 60° C. and 65° C.

Table 2 illustrates the general starch gelatinization temperature rangesfor a number of starches. The table has been complied from varioussources and is not meant to limit the invention, but is provided as aguide.

TABLE 2 Temperature Range for the Gelatinization of StarchesGelatinization Starch Temperature Range ° C. Barley 52-59 Wheat 58-64Rye 57-70 Corn (maize) 62-72 High amylose corn 67-80 Rice 68-77 Sorghum68-77 Potato 58-68 Tapioca 59-69 Sweet Potato 58-72 (J. J. M. Swinkelspg 32-38 in STARCH CONVERSION TECHNOLOGY, Eds Van Beynum et al., (1985)Marcel Dekker Inc. New York and The Alcohol Textbook 3^(rd) ED. Areference for the beverage, fuel and industrial alcohol industries, EdsJacques et al., (1999) Nottingham University Press, UK)

The pH range at which the methods of the invention is conducted is inthe range of about pH 3.0 to pH 6.5; also the range of pH 3.5 to pH 6.5;the range of pH 4.0 to pH 6.5; and the range of pH 4.5 to pH 6.0 areused in the methods. The pH range is somewhat dependent of the specificenzymes and one skilled in the art would be able to determine the bestpH range for conducting the methods without undue experimentation. Insome embodiments, when corn is the substrate the pH range is about pH4.5 to pH 6.0 and also about pH 5.0 to pH 5.5.

In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 94%, 95%, 96%, 97%, 98% and 99% of the dry solids of the granularstarch is converted into a composition of glucose syrup. In someembodiments, the granular starch substrate is completely hydrolyzed. Incertain embodiments, at least 90% of the granular starch substrate ishydrolyzed in a time period of 24 hours. In other embodiments, at least95% of the granular starch substrate is hydrolyzed in a time period of24 hours. In other embodiments, the dextrose syrup produced according tothe invention will be about 32 to 46% ds syrup containing at least 90%glucose.

In some embodiments, the period of time required to hydrolyze thegranular starch to produce a glucose syrup is from about 2 to 100 hours.In some embodiments, the period of time is about 5 to 100 hours. Inother embodiments, the period of time is from about 10 to 100 hours. Instill other embodiments, the period of time is from 5 to 50 hours. Inother embodiments, the period of time is at least about 10 hours butless than about 50 hours. In preferred embodiments, the one-step processwill be conducted from 2 to 100 hours and in some embodiments, theprocess will be conducted from 5 hours to 50 hours.

Preferably, the yield of glucose in the solubilized composition (glucosepercent of the total solubilized dry solids) is at least about 85%, 90%,91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%and 99.5%. More preferably, the yield is at least about 95% and mostpreferably, the yield is at least about 96%.

The exact amounts of the components encompassed by the composition andmethods of the invention depend on the combination of enzymes applied aswell as the type of granular starch processed. In some embodiments, theratio of alpha amylase units to GSHE units (alpha amylase:GSHE) will bein the range of 15:1 to 1:15 and in some embodiments in the range of10:1 to 1:10. In other embodiments, the ratio will be in the range of5:1 to 1:5 and in further embodiments, the alpha amylase:GSHE will be4:1 to 1:4. In preferred embodiments, the ratio will be about 2:1 to 1:4and most preferably about 2:1 to 1:2.

The one-step process encompassed by the invention can include theaddition of further ingredients without reducing the effectiveness ofthe hydrolysis of granular starch. These further ingredients include butare not limited to other enzymes, such as cellulases, proteases,pullulanases, hemicellulase, xylanases and the like.

The glucose produced according to the method of the invention may beseparated from the reaction mixture by methods known in the art. Some ofthese methods include centrifugation, conventional filtration methodsand preferably membrane separation processes. Also mentioned is the useof ultrafiltration membrane systems. In one preferred embodiment, theglucose syrup is separated by filtration using a molecular weightcut-off (MWCO) ultrafiltration membrane. These membranes are known inthe art. In some embodiments, the membrane could be 1,000 to 1,000,000MWCO. In other embodiments, the separation membrane may be a 0.1 to 1.0microfilter type membrane.

Further Conversion of Glucose to Desired End Products—

In a method encompassed by the invention, glucose syrup is the preferredend product. However, the glucose may be further purified to yieldcrystalline dextrose by known methods.

The glucose may also be converted to other desired end products.Conversion of glucose to other desired end products may be accomplishedby any suitable method such as, enzymatic or chemical methods. In oneembodiment, conversion is accomplished by bioconversion of glucose bycontacting glucose obtained according to the invention with amicroorganism capable of converting the glucose to an end product. Thecontacting step may be a sequential step, wherein the glucose syrupproduced by the method of the invention is then contacted with amicroorganism to produce an end product, or the contacting step may be asimultaneous step, wherein the granular starch substrate is contactedwith the GSHE and alpha amylase enzyme in combination with amicroorganism capable of converting the glucose syrup produced by theenzyme conversion to an end-product. The microorganism may be awild-type, mutated or recombinant microorganism. In some embodiments,the desired end products are fructose, ascorbic acid (ASA)intermediates, such as gluconate, 2-keto-D-gluconate,2,5-diketo-D-gluconate, 2-keto-L-gulonic acid, idonic acid, erythorbicacid and ascorbic acid; ethanol, 1,3-propanediol, monosodium glutamate,amino acids, sugars alcohols, organic acids, and indigo.

When fructose is the desired end-product, the glucose syrup obtainedaccording to the present invention may be enzymatically converted to afructose syrup by a glucose isomerase. In some embodiments, the glucoseisomerase is immobilized. Contemplated glucose isomerases include thosecommercially available such as G ZYME™ G993 liquid and GENSWEET™(Genencor International, Inc.) and SWEETZYME T (Novozyme). (See, e.g.U.S. Pat. No. 3,939,041 and U.S. Pat. No. 4,687,742).

When ASA intermediates and ASA are the desired end-products, the glucosesyrup obtained according to the present invention may be enzymaticallybioconverted to gluconate using for example, glucose dehydrogenase (orglucose oxidase-catalase enzymes. Gluconate may be oxidized to2,5-diketo-D-gluconate (DKG) by a DKG reductase. DKG may be reduced to2-keto-L-gulonic acid (KLG) by a KLG reductase. KLG may then beconverted to ASA. Methods for converting glucose to ASA and ASAintermediates are well known (See, for example U.S. Pat. No. 4,945,052,U.S. Pat. No. 5,008,193; U.S. Pat. No. 5,817,490 and U.S. Pat. No.6,358,715).

When 1,3-propanediol is the desired end-product, glucose obtainedaccording to the invention may be contacted with E. coli or otherrecombinant microorganisms (See, for example U.S. Pat. No. 6,013,494,U.S. Pat. No. 5,356,812).

When ethanol is the desired end-product, glucose may be contacted eithersequentially or simultaneously with an ethanolgenic microorganism, suchas the yeast Saccharomyces cerevisiae to obtain ethanol. (See, forexample U.S. Pat. No. 4,316,956). Further examples of ethanolgenicmicroorganisms, which can be used in the methods of the invention, arethose expressing alcohol dehydrogenase and pyruvate decarboxylase suchas Zymomonas mobilis (See, for example U.S. Pat. Nos. 5,028,539;5,424,202; 5,487,989 and 5,514,583). Upon completion of the fermentationwith yeast, the ethanol may be recovered, for example by distillation,and used for potable, fuel and industrial ethanol products. By-productsof the fermentation include both liquid and solid material that can beseparated and further used. For example, the recovered solid material,such as distiller's dried grain (DDG) and the mixture of DDS with liquidby-products to form distiller's dried grain with solubles (DDGS) may beused as an animal feed. The use of yeast for the production of ethanolduring fermentation and ethanol production is further discussed in THEALCOHOL TEXTBOOK, A REFERENCE FOR THE BEVERAGE, FUEL AND INDUSTRIALALCOHOL INDUSTRIES, 3^(rd) Edition, Eds K. A. Jacques et al., 1999,Nottingham University Press, UK.

In some embodiments of the invention, when the glucose syrup isseparated from the reaction mixture by for example, centrifugation orfiltration as mentioned above, the remaining composition will includeresidual starch. The residual starch by-product may be used in variousapplications. For example, residual starch may be recycled and used as acomponent in a method according to the invention; the residual starchmay be used as a carbon feedstock in further fermentations; the residualstarch may be used in a conventional starch hydrolysis process; and theresidual starch may be used as an ingredient for food formulations. Onepreferred embodiment of the invention comprises simultaneouslycontacting a granular starch substrate with a GSHE and an alpha amylaseat a temperature below the gelatinization of the granular starch tohydrolyze the granular starch to obtain a glucose syrup, separating theglucose syrup from the reaction mixture to obtain a glucose syrupcomponent and a by-product component which includes residual starch.

In some embodiments, according to the invention, when the residualstarch is recycled in a recycling step and used in the methodencompassed by the invention, the residual starch will be simultaneouslycontacted with a composition comprising a GSHE and an alpha amylase at atemperature below the gelatinization temperature of the granular starchsubstrate. The residual starch component may include enzymes that havebeen retained by the separation membrane and/or GSHE and alpha amylaseenzymes that are newly added to the reactor. In some embodiments, therecycling step in combination with the simultaneous contacting step maybe repeated numerous times and further may take place under continuousrecycling conditions wherein the glucose syrup is separated by meansknown in the art. The contacting time of the various components in areactor or vessel would be the same as outlined above that is from 2 to100 hours. In some preferred embodiments, the residence time would bebetween 5 and 50 hours.

In the recycling step embodiment, the residual starch may be recycled toobtain glucose syrup. In one non-limiting example, a granular starchslurry (i.e. a corn starch slurry having 38-42% ds) may be hydrolyzedwith a Humicola GSHE (i.e., 1.0 GSHE U/g) and SPEZYME ethyl (i.e., 0.6AU/g) at a temperature of about 58-62° C. and a pH of 5.0 to 6.0 for20-24 hours, wherein at least 55% of the corn starch is hydrolyzed toproduce a glucose syrup having at least 90% glucose. The residual starchmay be recovered and resuspended and combined with a second round ofGSHE and alpha amylase. In the second round, approximately at least 90%of the starch is hydrolyzed yielding at least 90% glucose. The glucosesyrup may then be evaporated by means known in the art, such as byvacuum and then used as a glucose feed.

In the recycling step embodiment, the residual starch may be recycled toobtain end products other than glucose. For example, when the endproduct is ethanol, the granular starch substrate is contacted eitherseparately and sequentially or simultaneously with GSHE, alpha amylaseand an ethanolgenic organism to both hydrolyze the granular starch andproduce ethanol, the ethanol may be recovered by distillation and theremaining material which includes both solid and liquid materialincluding residual starch may be recycled and used with the GSHE andalpha amylase in further steps such that the recycling takes place undercontinuous recycling conditions.

EXPERIMENTAL

In the disclosure and experimental section which follows, the followingabbreviations apply: rH-GSHE (Humicola grisea var. thermoidea GSHEexpressed in Trichoderma reesei); wt % (weight percent); ° C. (degreesCentigrade); rpm (revolutions per minute); H₂O (water); dH₂O (deionizedwater); by (base pair); kb (kilobase pair); kD (kilodaltons); gm(grams); μg (micrograms); mg (milligrams); ng (nanograms); μl(microliters); ml and mL (milliliters); mm (millimeters); nm(nanometers); μm (micrometer); M (molar); mM (millimolar); μM(micromolar); U (units); V (volts); MW (molecular weight); sec(seconds); min(s) (minute/minutes); hr(s) (hour/hours); PAGE(polyacrylamide gel electrophoresis); Di (deionized); phthalate buffer(sodium phthalate in water, 20 mM, pH 5.0); Cerestar (Cerestar, Inc., aCargill Inc., Minneapolis, Minn.); AVICELL® (FMC Corporation); SDS(sodium dodecyl sulfate); Tris (tris(hydroxymethyl)aminomethane); w/v(weight to volume); v/v (volume to volume); Genencor (GenencorInternational, Inc., Palo Alto, Calif.); Shin Nihon (Shin Nihon, Japan).

General Methods:

Starch Substrates—Purified and/or refined cornstarch, wheat starch andtapioca starch were used in the examples provided below.

Oligosaccharides Analysis—The composition of the reaction products ofoligosaccharides was measured by high pressure liquid chromatographicmethod (Beckman System Gold 32 Karat Fullerton, Calif., USA) equippedwith a HPLC column (Rezex 8 u8% H, Monosaccharides), maintained at 50°C. fitted with a refractive index (RI) detector (ERC-7515A, RI Detectorfrom The Anspec Company, Inc.). Dilute sulfuric acid (0.01 N) was usedas the mobile phase at a flow rate of 0.6 ml per minute. Twentymicroliter of 4.0% solution was injected on to the column. The columnseparates based on the molecular weight of the saccharides. For examplea designation of DP1 is a monosaccahride, such as glucose; a designationof DP2 is a disaccharide, such as maltose; a designation of DP3 is atrisaccharide, such as maltotriose and the designation “DP4⁺” is anoligosaccharide having a degree of polymerization (DP) of 4 or greater.

Relative solubilization of the solids—A conventional low temperature jetcooking process was used to solubilize the starch (U.S. Pat. No.3,912,590). The measured BRIX was taken as 100% solubilization of thestarch under the defined parameters of starch to water ratio. In atypical jet cooking process, suspending 150 grams of starch in 350 gramsof water made a 30% starch slurry. The pH was then adjusted to pH 5.8using 10% NaOH. Thermostable alpha amylase, SPEZYME FRED (GenencorInternational Inc.) was added at 0.4 Kg/MT, ds and heated in a jetcooker maintained at 105° C. for 8 min. The gelatinized starch wasfurther hydrolyzed at 95° C. for 90 min. An aliquot of the hydrolysatewas withdrawn and centrifuged. The clear supernatant was used to measurethe BRIX (ABBE Refractometer, American Optical Corporation, ScientificInstrument Division, Buffalo, N.Y.). The BRIX for 100% solubilizedstarch for different starch substrate at 30% ds is given in Table 3 andused to calculate the percent relative solubilization of starch underdifferent treatment conditions. Alternatively, BRIX for 100%solubilization under different conditions was determined by incubating 5ml of an aliquot with 10 micro liter of SPEZYME FRED (GenencorInternational Inc) at 95° C. for 5 min. The high temperature treatedsample was kept at 85° C. for 2 hours. The insoluble solids wereseparated by centrifugation and the BRIX of the clear supernatant wasmeasured.

TABLE 3 BRIX For Enzyme jet cooked starch substrate at 30% slurry Enzymejet cooked Starch substrate, 30% ds Measured BRIX Cornstarch 28.2 Wheatstarch 27.9 Tapioca starch 28.5

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.Indeed, it is contemplated that these teachings will find use in furtheroptimizing the process systems described herein.

Example 1 Expression of Humicola grisea var. thermoidea GSHE Gene inTrichoderma reesei

A. Cloning of the Humicola grisea var. thermoidea GSHE Gene

Genomic DNA (SEQ ID NO:1) was extracted from frozen Scytalidiumthermophilum (ATCC 16453, anamorph, H. grisea var. thermoidea) mycelia.The frozen mycelia were ground with dry ice in a coffee grinder and theDNA was extracted by the EasyDNA protocol (Invitrogen). An extrachloroform/phenol/isoamyl alcohol extraction was added to the standardprotocol. PCR primers were designed, based on the NCBI databaseaccession #M89475 sequence. The forward primer contained a motif fordirectional cloning into the pENTR/D vector (Invitrogen).

The sequence of the RSH003f primer was CAACATGCATACCTTCTCCAAGCTCCTC (SEQID NO. 7) and the sequence of the RSHOO4r primer wasTTAACGCCACGAATCATTCA CCGTC (SEQ ID NO. 8).

The PCR product was cloned into pENTR/D, according to the InvitrogenGateway system protocol. The vector was then transformed into chemicallycompetent Tol10 E. coli (Invitrogen) with kanamycin selection. PlasmidDNA from several clones was restriction digested to confirm the correctsize insert. The gla1 insert was sequenced (Sequetech, Mountain View,Calif.) from several clones. Plasmid DNA from one clone, pENTR/D_N13,was added to the LR clonase reaction (Invitrogen Gateway system) withpTrex3g/amdS destination vector DNA. Recombination, in the LR clonasereaction, replaced the CmR and ccdB genes of the destination vector withthe H. grisea gla1 from the pENTR/D vector. This recombinationdirectionally inserted gla1 between the cbhl promoter and terminator ofthe destination vector. Recombination site sequences of 48 and 50 bpremained upstream and downstream, respectively, of gla1. An aliquot ofthe LR clonase reaction was transformed into chemically competent Top10E. coli and grown overnight with carbenicillin selection. Plasmid DNA,from several clones, was digested with appropriate restriction enzymesto confirm the correct insert size. Plasmid DNA from clone, pTrex3g_N13(see FIGS. 3 and 4) was digested with Xba1 to release the expressioncassette including the cbhl promoter:gla1:cbhl terminator:amdS. This 6.6kb cassette was purified by agarose gel extraction using standardtechniques and transformed into a strain of T. reesei derived from thepublicly available strain QM6a, as further described below.

The cassette was sequenced by Sequetech, Mountain View, Calif. and theDNA for GSHE is illustrated in FIG. 1 (SEQ ID NO:1) and the amino acidsequence illustrated in FIG. 2 (SEQ ID NOs:2 and 3).

B. Transformation of T. reesei—

Approximately 2 cm² of a plate of sporulated mycelia (grown on a PDAplate for 5 days at 30° C.) was inoculated into 50 ml of YEG (5 g/Lyeast extract plus 20 g/L glucose) broth in a 250 ml, 4-baffle shakeflask and incubated at 37° C. for 16-20 hours at 200 rpm. The myceliawere recovered by transferring the liquid volume into 50 ml conicaltubes and spinning at 2500 rpm for 10 minutes. The supernatant wasdecanted. The mycelial pellet was transferred into a 250 ml, 0.22 micronCA Corning filter bottle containing 40 ml of filtered β-D-glucanasesolution and incubated at 30° C., 200 rpm for 2 hrs to generateprotoplasts for transformation.

Protoplasts were harvested by filtration through sterile miracloth intoa 50 ml conical tube. They were pelleted by spinning at 2000 rpm for 5minutes and aspirated. The protoplast pellet was washed once with 50 mlof 1.2 M sorbitol, spun down, aspirated, and washed with 25 ml ofsorbitol/CaCl₂. Protoplasts were counted and then pelleted at 2000 rpmfor 5 min, the supernate was decanted, and the protoplast pellet wasresuspended in an amount of sorbitol/CaCl₂ sufficient to generate aprotoplast concentration of 1.25×10⁸ protoplasts per ml, generating aprotoplast solution.

Aliquots of up to 20 μg of expression vector DNA (in a volume no greaterthan 20 μl) were placed into 15 ml conical tubes and the tubes were puton ice. Then 200 μl of the protoplast suspension was added along with 50μl PEG solution to each transformation aliquot. The tubes were mixedgently and incubated on ice for 20 min. PEG solution (2 ml) was added tothe transformation aliquot tubes, and these were incubated at roomtemperature for 5 minutes. Sorbitol/CaCl₂ (4 ml) solution was added tothe tubes (generating a total volume of 6.2 ml). The transformationmixture was divided into 3 aliquots each containing about 2 ml. Anoverlay mixture was created by adding each of these three aliquots tothree tubes of melted top agar (kept molten by holding at 50° C.) andthis overlay mixture was poured onto a transformation plate. Thetransformation plates were then incubated at 30° C. for four to sevendays.

The transformation was performed with amdS selection. Acetamide/sorbitolplates and top agar were used for the transformation. Top agar wasprepared by the same Sorbitol/acetamide agar recipe as the plates,except that low melting agarose was used in place of Noble agar.Transformants were purified by transfer of isolated colonies to freshselective media containing acetamide (i.e., Sorbitol/acetamide agar,without sorbitol).

With reference to the examples the solutions were prepared as follows.

-   -   1) 40 ml β-D-glucanase solution was made up in 1.2M sorbitol and        included 600 mg β-D-glucanase (InterSpex Products Inc., San        Mateo, Calif.) and 400 mg MgSO₄.7H₂O.    -   2) 200 ml PEG mix contained 50 g PEG 4000 (BDH Laboratory        Supplies Poole, England) and 1.47 g CaCl₂.2H₂O made up in dH₂O.    -   3) Sorbitol/CaCl₂ contained 1.2M sorbitol and 50 mM CaCl₂.    -   4) Acetamide/sorbitol agar:        -   Part 1—0.6 g acetamide (Aldrich, 99% sublime), 1.68 g CsCl,            20 g glucose, 20 g KH₂PO₄, 0.6 g MgSO₄.7H₂O, 0.6 g            CaCl₂.2H₂O, 1 ml 1000× salts (see below), adjusted to pH            5.5, brought to volume (300 mls) with dH₂O, filter            sterilized.        -   Part II—20 g Noble agar and 218 g sorbitol brought to volume            (700 mls) with dH₂O and autoclaved.        -   Part II was added to part I for a final volume of 1 L.    -   5) 1000× Salts—5 g FeSO₄.7H₂O, 1.6 g MnSO₄.H₂O, 1.4 g        ZnSO₄.7H₂O, 1 g CoCl₂.6H₂O were combined and the volume was        brought to 1 L with dH₂O. The solution was filter sterilized.        C. Fermentation of T. reesei Transformed with the H. grisea var.        thermoidea GSHE Gene.

In general, the fermentation protocol as described in Foreman et al.(Foreman et al. (2003) J. Biol. Chem. 278:31988-31997) was followed.More specifically, duplicate fermentations were run for each of thestrains displayed in FIG. 5. 0.8 L of Vogels minimal medium (Davis etal., (1970) Methods in Enzymology 17A, pg 79-143 and Davis, Rowland,NEUROSPORA, CONTRIBUTIONS OF A MODEL ORGANISM, Oxford University Press,(2000)) containing 5% glucose was inoculated with 1.5 ml frozen sporesuspension. After 48 hours, each culture was transferred to 6.2 L of thesame medium in a 14 L Biolafitte fermenter. The fermenter was run at 25°C., 750 RPM and 8 standard liters per minute airflow. One hour after theinitial glucose was exhausted, a 25% (w/w) lactose feed was started andfed in a carbon limiting fashion to prevent lactose accumulation. Theconcentrations of glucose and lactose were monitored using a glucoseoxidase assay kit or a glucose hexokinase assay kit withbeta-galactosidase added to cleave lactose, respectively(Instrumentation Laboratory Co., Lexington, Mass.). Samples wereobtained at regular intervals to monitor the progress of thefermentation. Collected samples were spun in a 50 ml centrifuge tube at¾ speed in an International Equipment Company (Needham Heights, Mass.)clinical centrifuge.

Sample supernatants were run of 4-12% BIS-TRIS SDS-PAGE gels, underreducing conditions with MOPS (morpholinepropanesulfonic acid) SDSrunning buffer and LDS sample buffer. The results are provided in FIG.5. Lanes 3, 4 and 5 illustrate a 68 kD rGSHE band at different timeperiods.

D. Assay of GSHE Activity from Transformed Trichoderma reesei Clones—

Enzyme activity—GSHE activity was determined as milligrams (mg) ofreducing sugars released (measured as glucose equivalent) per minute(min) during an incubation of 5 ml of 10% granular cornstarch in a 0.1 Macetate buffer, pH 4.5, 50° C. with an aliquot of the enzymepreparation. One unit of GSHE is defined as 1.0 mg of reducing sugarreleased per min under the assay conditions.

Native GSHE (nGSHE) from Humicola grisea var. thermoidea and recombinantGSHE produced from T. reesei were purified by standard techniques usinghydrophobic interaction chromatography using phenyl-Sepharose (AmershamBiosciences, Piscataway, N.J.) followed by ion exchange chromatographyusing SP-sepharose (Amersham Biosciences, Piscataway, N.J.). Therecombinant GSHE initially expressed by T. reesei clones included twoprotein peak fractions in about equal concentrations. These peaks werelabeled rGSHE1 and rGSHE2. The two peaks differed in mass by 1500D andby 0.3 pH units as measured by matrix assisted laser desorption andionization (MALDI-TOF) on a voyageur mass spectrometer (AppliedBiosystems, Foster City, Calif.) and an isoelectric focusing gel (SERVAElectrophoresis, GmbH, Heidelberg, Germany) according to manufacturerdirections. Both rGSHE1 and rGSHE2 have the same specific activity asmeasured by the raw starch hydrolyzing assay and protein measurementsusing a MicroBCA protein assay kit (Pierce, Rockford, Ill.) and thepercent solution extinction coefficient (A280 0.1%=1.963). After aperiod of time, measured at approximately 72 hours after initial rGSHEexpression, only one form of rGSHE is represented (rGSHE3). (See, Table4).

TABLE 4 Specific Activity Source of GSHE GSHE Units/mg % totalcarbohydrate Native GSHE 9.0 1.12 rGSHE1/rGSHE2 8.0/8.0 2.70 rGSHE3 8.00.57The % carbohydrate (CHO) of the GSHEs was determined by acid hydrolysisusing 4N trifluoroacetic acid at 100° C. for 5 hrs and measurements weremade of the released reducing sugars using parahydroxybenzoic acidhydrazide.

When initially expressed, the glycosylation of rGSHE1 and rGSHE2 was2.70% of the total carbohydrate. However, after 72 hours, the level ofglycosylation of rGSHE3 found in the medium was 0.57% total CHO. Thelevel of glycosylation of native GSHE was 1.12%.

E. Comparison of Native GSHE from H. grisea var. thermoidea andRecombinantly Expressed H. grisea var. thermoidea GSHE in Trichodermareesei.

(1) pH Stability was Determined from pH 3 to 7.

The collected samples of recombinantly produced GSHE as described aboveand samples of native GSHE were diluted to equal protein concentrationswith 20 mM acetate buffer at pH 4.5. Reactions were then run in 100 mMcitrate/NaOH buffers at 50° C. for 30 minutes at pH levels 3 to 7.

1.0 ml of the reaction was then added to 5 ml of 10% corn starch(Cargill Foods, Minneapolis, Minn.) in 100 mM acetate, pH 4.5 in sampletubes. The tubes were shaken at 50° C. for 20 minutes. Then 0.5 ml 2.0%NaOH was added. Tubes were spun and 0.5 ml of the supernatant wasassayed for reducing sugars using the Dinitro Salicylic acid (DNS) assay(Goto et al., (1994) supra).

The results of the assay are depicted in FIG. 8A. The recombinantlyproduced GSHE exhibited about 80% residual activity at pH 3.5. Incomparison, the corresponding native GSHE exhibited only about 20%residual activity. At pH 4.0 both the recombinantly produced GSHE andthe native GSHE exhibited about 82% residual activity and at pH 5.5 bothenzymes exhibited between about 90 to 100% residual activity.

Stability was also measured at pH 7.5 to 10.5 using the methods asdescribed above. However, the buffer was 100 mM boric acid/NaOH buffer.As exhibited in FIG. 8B, at pH 7.5 both enzymes exhibited about 100%residual activity. At pH 8.5 recombinantly produced GSHE exhibited about82% residual activity and the native GSHE exhibited about 90% residualactivity. At pH 9.5 the % residual activity of recombinantly producedGSHE was substantially less than the native GSHE. (10% compared to 72%,respectively).

(2) Profile of Activity as a Function of Temperature.

Temperature stability was determined at pH 5.75. Using essentially thesame procedures as described above for the pH stability studies, enzymesamples were diluted to equal protein concentrations in a 100 mM acetatebuffer and then 1.0 ml of the diluted enzymes was exposed to a waterbath temperature of 40° C., 50° C., 60° C. and 70° C. for 10 minutes andassayed as described above in the pH stability studies. The results arepresented in Table 5.

TABLE 5 Temp % GSHE Source ° C. Residual Activity Native GSHE 40 100 5095 60 90 70 0 Recombinant GSHE 40 100 50 93 60 92 70 0 % residualactivity means the % difference referenced to 100% at pH 4.0

The profile of activity as a function of temperature of therecombinantly produced GSHE is similar to that of the correspondingnative GSHE.

(3). Hydrolysis of Granular Corn Starch by nGSHE and rGSHE.

Both native GSHE from H. grisea var. thermoidea (nGSHE) andrecombinantly expressed H. grisea var. thermoidea (rGSHE) in Trichodermareesei were diluted to equal protein concentrations in pH 4.5 acetatebuffer. One ml of the dilution was added to a 10% corn starch (CargillFoods, Minneapolis, Minn.) slurry in 20 mM pH 4.5 acetate buffer andshaken at 350 rpm at 50° C. At designated time intervals 100 μL ofslurry was removed and added to 10 μL of 2% NaOH. The sample was spunand the supernatant was assayed for glucose (mg glucose/mg protein)using the glucose oxidase reagent in a Monarch clinical analyzer(Instrumentation Laboratory, Lexington, Mass.). As shown in FIG. 9 thehydrolysis of corn starch was slightly lower for the rGSHE compared tothe nGSHE.

Example 2 Expression of Aspergillus awamori var. kawachi GSHE Gene inTrichoderma reesei

A. Cloning the Aspergillus awamori var. kawachi GSHE Gene

Genomic DNA was extracted from frozen mycelia of a strain of A. awamorivar. kawachi according to the methods described in Example 1. The PCRprimer sequences were designed based on the published sequence of the A.awamori var. kawachi glucoamylase GAI (Hayashida, et al. (1989) Agric.Biol. Chem. 53:923-929). This GAI is a GSHE. The following primers wereused: the RSH10f primer having the sequence, CAC CAT GTC GTT CCG ATC TCTTCT C (SEQ ID NO:9), which includes the Gateway (Invitrogen) directionalcloning motif CACC and the RSH11r primer having the sequence, CTA CCGCCA GGT GTC GGT CAC (SEQ ID NO:10).

The DNA sequence is provided in FIG. 6 (SEQ ID NO:4). The encoded GSHEpolypeptide sequence, including the signal peptide, is provided in FIG.7A (SEQ ID NO:5) and the mature protein sequence is provided in FIG. 7B(SEQ ID NO:6).

The 2.16 kb PCR product was gel-purified (Gel Purification kit, Qiagen)and cloned into pENTR/D (Invitrogen), according to the Gateway systemprotocol. The vector was then transformed into chemically competentTop10 E. coli (Invitrogen) with kanamycin selection. Plasmid DNA fromseveral clones was restriction digested to confirm the correct sizeinsert. The GAI gene insert was sequenced (Sequetech, Mountain View,Calif.) from several clones (SEQ ID NO:4). Plasmid DNA from one clone,pENTR/D_Ak33xx#1, was added to the LR clonase reaction (InvitrogenGateway system) with the pTrex3g/amdS destination vector DNA.Recombination, in the LR clonase reaction, replaced the Cm^(R) and ccdBgenes of the destination vector with the A. kawachi GAI from the pENTR/Dvector. This recombination directionally inserted GAI between the cbhlpromoter and terminator of the destination vector. AttB recombinationsite sequences of 48 and 50 bp remained upstream and downstream,respectively, of the glucoamylase. Reference is made to FIG. 3, whereinthe H. grisea gla1 has been replaced by the A. kawachi GAI in thisexample. Two microliters of the LR clonase reaction were transformedinto chemically competent Top10 E. coli and grown overnight withcarbenicillin selection. Plasmid DNA from several clones was digestedwith XbaI to confirm the insert size. Plasmid DNA from clone,pTrex3g_Akxx #3 was digested with XbaI to release the expressioncassette including the cbh/promoter:GAI:cbhl terminator:amdS. This 6.7kb cassette was purified by agarose extraction using standard techniquesand transformed into a strain of T. reesei derived from the publiclyavailable strain QM6a.

B. Transformation of T. reesei with the A. awamori var. kawachi GSHEGene

A Trichoderma reesei spore suspension was spread onto the center ˜6 cmdiameter of an MABA transformation plate (150 μl of a 5×10⁷-5×10⁸spore/ml suspension). The plate was then air dried in a biological hood.Stopping screens (BioRad 165-2336) and macrocarrier holders (BioRad1652322) were soaked in 70% ethanol and air dried. DriRite desiccant wasplaced in small Petri dishes (6 cm Pyrex) and overlaid with Whatmanfilter paper. The macrocarrier holder containing the macrocarrier(BioRad 165-2335) was placed flatly on top of filter paper and the Petridish lid replaced.

A tungsten particle suspension was prepared by adding 60 mg tungstenM-10 particles (microcarrier, 0.7 micron, Biorad #1652266) to anEppendorf tube. One ml ethanol (100%) was added. The tungsten wasvortexed in the ethanol solution and allowed to soak for 15 minutes. TheEppendorf tube was microfuged briefly at maximum speed to pellet thetungsten. The ethanol was decanted and washed three times with steriledistilled water. After the water wash was decanted the third time, thetungsten was resuspended in 1 ml of sterile 50% glycerol. The tungstenwas prepared fresh every two weeks.

The transformation reaction was prepared by adding 25 μl of suspendedtungsten to a 1.5 ml Eppendorf tube for each transformation. Subsequentadditions were made in order, 0.5-5 μl DNA (0.2-1 μg/μl), 25 μl 2.5MCaCl₂, 10 μl 0.1 M spermidine. The reaction was vortexed continuouslyfor 5-10 minutes, keeping the tungsten suspended. The Eppendorf tube wasthen microfuged briefly and decanted. The tungsten pellet was washedwith 200 μl of 70% ethanol, microfuged briefly to pellet and decanted.The pellet was washed with 200 μl of 100% ethanol, microfuged briefly topellet, and decanted. The tungsten pellet was resuspended, by pipetting,in 24 μl 100% ethanol. The Eppendorf tube was placed in an ultrasonicwater bath for 15 seconds and 8 μl aliquots were transferred onto thecenter of the desiccated macrocarriers. The macrocarriers were left todry in the desiccated Petri dishes.

A He tank was turned on to 1500 psi. 1100 psi rupture discs (BioRad165-2329) were used in the Model PDS-1000/He Biolistic Particle DeliverySystem (BioRad). When the tungsten solution was dry, a stopping screenand the macrocarrier holder were inserted into the PDS-1000. An MABAplate, containing the target T. reesei spores, was placed 6 cm below thestopping screen. A vacuum of 29 inches Hg was pulled on the chamber andheld. The He Biolistic Particle Delivery System was fired. The chamberwas vented and the MABA plate removed for incubation, 28° C. for 5-7days.

With reference to Example 2 the solutions were prepared as follows.

Modified amdS Biolistic agar (MABA) per liter Part I, make in 500 mldH₂O 1000x salts 1 ml Noble agar 20 g pH to 6.0, autoclave Part II, makein 500 ml dH₂O Acetamide 0.6 g CsCl 1.68 g Glucose 20 g KH₂PO₄ 15 gMgSO₄•7H₂O 0.6 g CaCl₂•2H₂O 0.6 g

-   -   pH to 4.5, 0.2 micron filter sterilize; leave in 50° C. oven to        warm, add to Part I, mix, pour plates.

1000x Salts per liter FeSO₄•7H₂O 5 g MnSO₄•H₂O 1.6 g ZnSO₄•7H₂O 1.4 gCoCl₂•6H₂O 1 g 0.2 micron filter sterilize

Expression of rGSHE (A. awamori var. kawachi GSHE expressed in T.reesei) was determined as described above for expression of H. griseavar. thermoidea in Example 1. The level of expression was determined tobe greater than 1 g/L (data not shown). FIG. 10 provides the results ofa SDS-PAGE gel illustrating the expression of Aspergillus awamori var.kawachi GSHE in the T. reesei host.

Example 3 Solubilization and Hydrolysis of Different Granular StarchSubstrates by Alpha Amylase

In a typical experiment, 150 grams of granular starch were suspended in350 grams of distilled water. After mixing, the pH was adjusted to pH5.5 using 6 N NaOH. The alpha amylase (GZYME G997 at 1.0 kg/MT ofstarch, ds) was added to the starch slurry and incubated with constantstirring in a water bath maintained at 60° C. The samples were withdrawnat different time intervals for measuring the Brix. The sample withdrawnat 24 hrs was used to determine the sugar composition using HPLC (Table6).

TABLE 6 G Zyme G 997 % Solids solublized % Carbohydrate composition (0.1kg/MT starch) Incubation time (hr) at 24 hr Starch substrate 2 4 6 9 1224 DPI DP2 DP3 DP4⁺ Corn 32.2 39.3 42.9 49.2 52.8 53.9 0.6 12.0 15.072.4 Tapioca 46.6 50.8 53.6 56.1 58.2 62.4 1.6 11.6 14.6 72.2 Wheat 74.980.6 82.4 84.5 86.3 87.8 1.3 11.5 14.1 73.1The results illustrated in Table 6 show significant differences in thesolubilization of the granular starch substrates. Wheat had the highest% of solubilized solids, and corn had the lowest percent. Significantdifferences were not observed in the sugar composition after 24 hours.

Example 4 Solubilization and Hydrolysis of Granular Starch Substrates byAlpha Amylase (G ZYME G 997) and rH-GSHE

In a typical experiment, 350 g of water was added separately to 150grams of each, granular cornstarch, granular wheat starch and granulartapioca starch and the pH was adjusted to pH 5.5 using 6N NaOH. Theslurry was kept in a water bath, which was maintained at 60° C. withcontinuous stirring for uniform mixing. After stabilization of thetemperature, alpha amylase as G Zyme G 997 (0.1 Kgs/MT of starch ds) andrH-GSHE (1.0 GSHE Units/gram of starch ds) were added to each starchslurry and incubation was continued. Samples were taken at differentintervals of time, centrifuged and Brix was checked. The 24 hour sampleswere analyzed for sugar composition. The relative solubilization of thegranular starch was calculated by comparing the Brix from the jetcooking process and reference is made to Table 7.

TABLE 7 % Relative Solubilization (hrs) % Soluble Sugar (24 hrs) Starch2 4 6 12 18 24 DP1 DP2 DP3 DP4⁺ Wheat 86.0 91.0 93.9 95.7 97.5 98.2 97.32.3 0.2 0.2 Corn 61.3 78.4 87.9 98.2 100.0 100.0 97.4 2.1 0.2 0.2Tapioca 64.9 75.4 81.8 87.4 91.6 97.5 96.5 1.6 0.4 1.5

The combined effect of G Zyme G 997 and rH-GSHE resulted in almostcomplete solubilization of the granular starch substrate under mildconditions compared to the current high temperature jet cooking process.The analysis of the 24-hour samples showed glucose yield greater than96.5%.

Example 5 Solubilization and Hydrolysis of Granular Cornstarch byGlucoamylases Having Granular Starch Hydrolyzing Activity

Commercially available glucoamylases exhibiting granular starchhydrolyzing activity from Aspergillus niger (OPTIDEX L-400 and G Zyme G990 4X from Genencor International Inc), and Rhizopus niveus (CU. CONC.from Shin Nihon Chemicals, Japan) were compared with rH-GSHE asdescribed above in example 1. The granular starch hydrolyzing activityof these products was measured using the assay described above.

TABLE 8 Glucoamylase GSHE units/g OPTIDEX L-400 555 G Zyme G 990 4X 474CU. CONC. 1542 rH-GSHE 518

In a typical experiment, a 30% granular cornstarch slurry in distilledwater (150 grams of starch in 350 grams of distilled water) was preparedand the pH was adjusted to pH 5.5 using 6 N NaOH. G Zyme G 997 was addedat 0.1 kg/MT of starch ds and the starch slurry was kept in a water bathmaintained at 60° C. To each starch slurry containing G Zyme G 997,different glucoamylases were added at an equal dosage, i.e.; 1.5 GSHEunits/gram starch, ds and incubated at 60° C. An aliquot was withdrawnat different time intervals and centrifuged. The clear supernatant wasused for measuring the Brix. The sample incubated for 2, 6, 22.5 and 49hours was analyzed by HPLC for total sugar composition and the resultsare shown in Table 9.

TABLE 9 % % % % % Enzyme Hr DP1 DP2 DP3 DP4+ Solublized Distillase L-4002 94.2 0.9 0.2 4.7 G990 4X 2 95.9 0.7 0.2 3.2 Cu Conc 2 73.5 11.1 1.414.0 rH-GSHE 2 96.4 1.1 0.1 2.3 Distillase L-400 6 96.1 1.2 0.2 2.5 G9904X 6 96.7 1.4 0.2 1.7 Cu Conc 6 79.1 8.0 1.0 11.8 rH-GSHE 6 97.9 1.4 0.00.7 Distillase L-400 22.5 96.8 2.1 0.2 0.9 G990 4X 22.5 96.6 2.5 0.2 0.7Cu Conc 22.5 81.9 6.5 1.1 10.5 rH-GSHE 22.5 96.2 3.4 0.2 0.1 DistillaseL-400 49 96.3 3.0 0.2 0.5 81.6 G990 4X 49 96.0 3.3 0.2 0.4 81.6 Cu Conc49 80.8 6.9 1.5 10.8 74.6 rH-GSHE 49 93.8 5.6 0.5 0.1 97.5 Glucoamylaseswere added at a dose of 1.5 GSHEU/g to a starting slurry having 0.1kG/MT ds of alpha amylase.

Example 6 The Effect of pH on the Solubilization of Granular Cornstarch(35% Slurry) During Incubation with Alpha Amylase (G Zyme G 997) andrH-GSHE

In a typical experiment, 372 grams of water was added to 178 grams ofcornstarch. The slurry was stirred well for uniform mixing and the pH ofthe slurry was adjusted to pH 4.0, 5.0, 5.5, 6.0, 6.5 and 7.0 using 6 NNaOH. The samples were then kept in a water bath maintained at 60° C.After equilibration of the temperature, Zyme G997 at 0.1 kg/MT starchand rH-GSHE as described in example 1 (1.0 GSHE Units/g starch) wereadded to the slurry. The slurry was continuously stirred duringincubation and samples were taken after one hour for measuring the brix(Table 10).

TABLE 10 Incubation pH at 60° C. % Maximum Solubilization 4.0 9.9 5.0100.0 5.5 100.0 6.0 95.0 6.5 92.0 7.0 76.7

The maximum solubilization occurred at pH 5.0 and pH 5.6. A significantreduction in the solubilization of the granular cornstarch occurredbelow pH 5.0 and pH 5.5 at 60° C. indicating either lower activity orinactivation of the enzymes.

Example 7 Effect of Temperature on the Solubilization of the GranularCornstarch (32% Slurry) During Incubation with Alpha Amylase and rH-GSHE

In a typical experiment, 372 grams of water was added to 178 grams ofcornstarch. The slurry was stirred well for uniform mixing, and the pHof the slurry was adjusted to pH 5.5, using 6 N NaOH. The samples werekept in a water bath maintained at 55° C., 60° C. and 65° C. Afterequilibration of the temperature, Zyme G997 at 0.1 Kgs/MT starch andrH-GSHE as described in example 1 (1.0 GSHE Units/g starch) were added.The slurry was continuously stirred during incubation and the brix wasmeasured after one hour. (Table 11).

TABLE 11 Incubation Temperature ° C. % Starch Solubilized 55 28.7 6051.4 65 59.6 70 75.1

The solubility of the granular cornstarch was increased with increasingtemperature in the presence of G Zyme G997 and rH-GSHE. However HPLCanalysis of the solubilized carbohydrate above 65° C. indicatedinactivation of rH-GSHE as evidenced by lower level of glucose content.The increase in the dissolved solids content at higher temperature (>65°C.) was mainly due to the liquefaction effect of G Zyme G997 on granularcornstarch at higher temperatures.

Example 8 Effect of G Zyme G997 and rH-GSHE Concentrations on theSolubilization and Hydrolysis of Granular Cornstarch

In different flasks granular cornstarch (178 g) in 372 g water wasstirred well for uniform mixing. The pH of the slurry was adjusted to pH5.5. Two different levels of G Zyme G 997, 0.1 Kgs/MT starch and 0.5Kgs/MT were incubated with rH-GSHE at 0.25. 0.5, and 1.0 GSHE units/g dsstarch at 60° C. Samples were drawn at different intervals time, andused for measuring the brix and total sugar composition (Tables 12A and12B).

TABLE 12A Enzyme Concentration G Zyme rH-GSHE G997 Kgs/ GSHE Units/ %Relative Solubilization MT starch g starch 3 hr 6 hr 9 hr 12 hr 24 hr 30hr 0.1 0.25 50.9 59.3 70.2 75.1 85.8 88.6 0.1 0.50 54.3 72.9 80.4 85.294.0 96.8 0.1 1.0 60.7 80.4 88.6 92.1 98.1 100 0.5 0.25 60.9 71.9 77.683.3 91.2 94.0 0.5 0.5 66.9 79.5 85.8 89.9 97.2 99.4 0.5 1.0 76.7 87.492.4 95.3 99.1 99.7

The results in Table 12A indicate that increasing the dosage of G Zyme G997 from 0.1 Kgs/MT of starch to 0.5 Kg/MT of starch resulted in afaster solubilization of granular starch. But at both levels greaterthan 95% solubilization of granular starch occurred in 24 hours inpresence of 1.0 GSHE units of rH-GSHE. The effect of rH-GSHEconcentration on the solubilization of granular starch in the presenceof G Zyme G 997 increased dramatically with increasing dosage ofconcentration. The above results clearly show that neither of theenzymes, G Zyme G 997 or rH-GSHE alone can solublize the granular starchto completion. However, complete solubilization of the granular starchoccurred when the enzymes were added together.

The carbohydrate (sugar) composition of the solubilized granular (32%slurry) cornstarch during incubation of the granular cornstarch with GZYME G997 and rH-GSHE at 12, 24 and 30 hour at pH 5.5 and 60° C. wasanalyzed by HPLC and reference is made to Table 12B.

TABLE 12B Enzyme composition Incubation % G Zyme G997 rH-GSHE Timestarch % Carbohydrate Composition kg/MT st GSHE U/g st (hr) solubilizedDP1 DP2 DP3 DP4⁺ 0.1 0.25 12 75.1 87.9 4.1 0.9 7.2 24 85.8 93.2 2.4 0.83.6 30 88.6 94.7 2.0 0.8 2.6 0.1 0.50 12 85.2 95.9 1.6 0.4 2.1 24 94.097.0 1.8 0.3 0.9 30 96.8 97.5 2.0 0.2 0.4 0.1 1.00 12 92.1 97.5 1.9 0.20.5 24 98.1 96.9 2.8 0.2 0.2 30 100.0 96.3 3.3 0.3 0.2 0.5 0.25 12 83.384.4 6.4 1.4 7.8 24 91.2 92.0 3.0 1.3 3.6 30 94.0 94.0 2.3 1.1 2.6 0.50.50 12 98.9 95.0 2.0 0.9 2.4 24 97.2 96.6 1.9 0.5 0.9 30 99.4 96.9 2.10.4 0.6 0.5 1.00 12 95.3 97.1 1.9 0.3 0.7 24 99.1 96.7 2.3 0.3 0.2 3099.7 96.4 3.2 0.3 0.2

The results in Table 12B illustrate that an appropriate blend ofBacillus stearothermophilus alpha amylase and rH-GSHE would meet avariety of demands in the commercial production of sugar sweeteners andbiochemicals directly from granular starch without applying conventionalhigh temperature cooking process. High levels of alpha amylaseaccelerates the rate of solubilization of granular starch but higherlevel of rH-GSHE resulted in high levels of reversion reaction productsresulting in significantly low levels of higher sugar.

Example 9 Comparison on the Hydrolysis of Enzyme Liquefied CornstarchSubstrate (Soluble Starch Substrate) and Granular Cornstarch Substrate(Insoluble) by Glucoamylase Preparations

In a typical experiment, cornstarch (32% ds) was liquefied at pH 5.6using the low temperature jet cooking process (105° C., 8 min) followedby hydrolysis at 95° C. for 90 min. SPEZYME FRED (Genencor InternationalInc) was added at 0.4 Kgs/MT of starch, ds. in the liquefaction process.The pH of the SPEZYME FRED liquefied starch substrate was adjusted to pH4.2 and glucoamylase (OPTIDEX L-400) was added at 0.22GAU/g ds. Thehydrolysis was carried out at 60° C. Samples were withdrawn at differenttime intervals and analyzed by HPLC to determine the time required forreaching the maximum glucose yield.

Thirty two percent ds granular cornstarch slurry in distilled water wasprepared and the pH of the slurry was adjusted to pH 5.5 using 1 N NaOH.The flask was then kept in a water bath maintained at 60° C. and G ZymeG997 was added at 0.1 Kgs/MT ds and rH-GSHE was added at 1.0 GSHEUnits/gram ds and the sample was incubated at 60° C. with constantstirring. Samples were withdrawn at different intervals of time formeasuring the BRIX and glucose yield (Table 13).

TABLE 13 Time (hr) for Composition at Maximum. Gluco- reaching Glucoseyield (%) Substrate amylase Max. glucose DP1 DP2 DP3 DP4⁺ LiquefiedOPTIDEX 61 95.2 3.1 0.4 1.3 Starch L-400 Soluble, 32% slurry GranularrH-GSHE 24 96.8 2.8 0.2 0.2 Starch and G Insoluble, ZYME G997 32% slurry

Hydrolysis of the liquefied soluble starch by glucoamylase required alonger time to achieve the maximum glucose yield compared to thehydrolysis of insoluble granular starch. At the peak time for reachingmaximum glucose yield, the glucose level by granular starch as comparedto liquefied soluble starch was higher with significantly lower sugarsat DP4⁺ (96.8 and 0.2 compared with 95.2 and 1.3).

Higher glucose yield, the potential for shorter saccharification timeand a total elimination of high temperature jet cooking step,differentiates the application of the alpha amylase and GSHE enzymeblend of the present invention.

Example 10 Effect of Granular Cornstarch Concentration on theSolubilization and Hydrolysis of Starch During the Incubation with GZyme G 997 (0.1 Kgs/MT) and rH-GSHE (1.0 GSHE Units/g).

Different concentrations of granular cornstarch slurry were prepared indistilled water. i.e., 32%, 35%, 38%, 40% and 42%. The pH of the slurrywas adjusted to pH 5.5. The samples were then kept in a water bathmaintained at 60° C. and stirred continuously for uniform mixing. G ZymeG997 (0.1 Kgs/MT of ds) and rH-GSHE obtained from Example 1 (1.0 GSHEunits/g ds) were added to the slurry. An aliquot sample was withdrawn atdifferent time intervals during incubation for measuring brix and sugarcomposition (Table 14).

TABLE 14 Starting Sample % Cabohydrate Profile Trial % DS % DS HoursDP > 3 DP3 DP2 DP1 1 32 0.41 0 1 32 21.31 2.5 1 32 26.84 7 1.56 0.351.71 96.38 1 32 33.55 24 0.28 0.39 3.30 96.03 1 32 34.50 48 0.49 0.403.83 95.28 2 35 0.49 0 2 35 22.57 2.5 2 35 28.57 7 1.24 0.35 1.81 96.602 35 34.50 24 0.33 0.71 4.16 94.80 2 35 36.91 48 0.21 0.68 4.71 94.40 338 0.62 0 3 38 23.89 2.5 3 38 29.67 7 1.38 0.33 1.84 96.45 3 38 35.76 240.31 0.38 3.21 96.11 3 38 38.64 48 0.48 0.56 4.76 94.19 4 40 0.34 0 4 4027.78 2.5 4 40 30.47 7 1.36 0.35 1.94 96.35 4 40 36.58 24 0.38 0.50 2.7996.33 4 40 39.71 48 0.22 0.70 4.77 94.31 5 42 0.48 0 5 42 25.31 2.5 5 4231.64 7 1.23 0.34 2.00 96.43 5 42 37.94 24 0.63 0.35 3.09 95.93 5 4240.82 48 0.40 1.29 6.57 91.74

The results show over 96% glucose syrup could be reached within 24 hoursof the saccharification at dissolved solids as high as 36%. The glucoseyield of greater than 96% at a solid level higher than 35% was reachedwhen an insoluble granular starch was used in the saccharificationprocess.

Example 11

As taught above and known in the art, the enzyme-enzyme starchconversion process for high glucose syrup consists of producing asoluble liquefact by subjecting an insoluble starch substrate to a hightemperature liquefaction process using a thermostable alpha amylase. Itis the normal practice in the commerce to inactivate the residual alphaamylase activity prior to the saccharification by glucoamylase to reducethe loss of glucose yield due to the presence of active alpha amylase.The inactivation of the residual alpha amylase activity is normallycarried out by decreasing the liquefact to pH 4.2 at 95° C. Hightemperature and pH 4.5 result in the complete inactivation of the alphaamylase. So we studied the effect of glucose yield with and withoutactive alpha amylase during saccharification of liquefact starch at pH5.5.

In a typical experiment, soluble liquefact from cornstarch was producedusing G Zyme G 997 (0.4 Kgs/MT starch) as a liquefying enzyme under jetcooking process conditions (32% ds starch at 105° C. at 8 min followedby 95° C. for 90 min). A portion of the liquefied starch was furtherheated to inactivate the residual alpha amylase activity. Thesaccharification of the liquefied starch with and without residual alphaamylase activity was further saccharified (32% ds) at pH 5.5, 60° C.using rH-GSHE as described in example 1 at 0.5 GSHE units/g. Sampleswere withdrawn at different intervals of time and analyzed for glucoseyield using HPLC (Table 15).

TABLE 15 Alpha Amylase Activity During Sac- Sac. Time % SugarComposition charification Substrate (Hrs) DP1 DP2 DP3 DP4⁺ InactiveSoluble 18 91.48 1.85 0.41 6.25 Liquefact 24 94.37 1.88 0.33 3.43 4296.08 2.74 0.32 0.86 48 96.01 2.95 0.30 0.74 68 95.25 3.79 0.44 0.52Active Soluble 18 91.88 2.54 1.19 4.39 Liquefact 24 94.31 2.20 1.00 2.4942 95.78 2.84 0.62 0.77 48 95.85 3.03 0.53 0.59 68 95.33 3.85 0.57 0.24Active Insoluble 24 96.84 2.75 0.23 0.18 starch

The results in Table 15 demonstrate that the presence of alpha amylaseactivity of G Zyme G 997 during the saccharification of the solublestarch substrate (liquefact) by glucoamylase resulted in the lowerglucose yield. Whereas, alpha amylase enhances the hydrolysis ofinsoluble (granular) starch substrate by glucoamylase resulting in asubstantially high level of glucose.

Example 12 Production of Glucose Syrup and Residual Starch fromHydrolysis of Corn Starch

In a reactor vessel, granular cornstarch (800 g) in 1200 g water wasstirred well for uniform mixing to obtain a slurry. The pH of the slurrywas adjusted to pH 5.5 with 4 N NaOH. G ZYME G 997 (0.1 Kgs/MT starch)and Humicola grisea var. theromidea expressed in Trichoderma reesei(rH-GSHE) at 1.0 GSHE U/g starch were added at 60° C. Samples werewithdrawn at various time intervals and used for measuring the brix andtotal sugar composition (Table 16).

TABLE 16 Reaction Time 3 hr 6 hr 9 hr % Solublization 60.7 80.4 88.6After achieving greater that 90% solubilization of granular starch in a10 hour reaction time, the sugar composition, measured by HPLC, of thesolubilized granular cornstarch was achieved as illustrated in Table 17.

TABLE 17 Sugar Type DP1 DP2 DP3 DP 4+ % Composition 97.5 1.9 0.2 0.5

After a 10 hr reaction time, the hydrolysis was stopped by adjusting thepH to 3.5 with 4NH₂SO₄. The syrup mixture contained greater than 96%dextrose at greater than 30% dissolved solids. The mixture was filteredat 60° C. by using a 500,000 molecular weight cut-off (MWCO)ultrafiltration membrane cartridge (AG Technology, MA). The residualstarch could then be recycled which would significantly reduced capitaland operating costs incurred in the production of glucose syrup.

A sample of the residual starch was dried and examined for its structureby scanning electron microscope (Hitachi S-5000 cold field emission SEM(Tokyo, Japan)) and compared with a typical starch granular beforeexpose to enzymes in accordance with the method of the invention (FIG.12). The dried samples were mounted on SEM sample stubs usingdouble-sided adhesive carbon tape. Any excess sample was removed bydusting with compressed air. The samples were then mounted at about a30° angle in a Bal-Tec Med 020 Modular High Vacuum Coating System(Liechtenstein) and sputter coated with 10 nn of platinum (Pt) whilerotating at 60 rpm. These settings ensured that the granules were evenlycoated on all sides. The thickness of the coating is negligible comparedto the size of the features resulting from the enzymatic action of thestarch granules. The accelerating voltage varied from 2-5 kV andmagnification was between 500-15,000×.

Micrograph FIG. 12 a depicts a typical starch granule before exposure toan enzyme composition and method of the invention. The surface is smoothand homogenous and the only noticeable feature is a fine cracking due tothe platinum metal coating. Once exposed to the enzyme blend and methodof the invention, the surface morphology of the granules change. As seenin micrographs b-d of FIG. 12, large round holes are bored into thegranules due to enzyme digestion of the starch granule substrate. Theholes range in diameter and vary in depth, and reflect a population ofstarch granules at different kinetic stages of enzymatic reaction. Somegranules have only a few number of holes and some are nearly covered inholes (micrograph b). Some granules were also sliced in half revealing across section of digested interior (micrographs c and d). Micrograph d)reveals granule digestion to completion, showing a fragment of ahollowed out shell.

1. A process for producing an amino acid end product from a granularstarch slurry, said process comprising: contacting a granular starchslurry obtained from a granular starch substrate simultaneously with analpha amylase and a glucoamylase, wherein the glucoamylase has at least97% amino acid sequence identity with SEQ ID NO:3, at a temperatureequal to or below the gelatinization temperature of the granular starch;allowing the alpha amylase and the glucoamylase to act for a period oftime sufficient to hydrolyze the granular starch comprising to obtainglucose; contacting the glucose with a microorganism capable ofconverting the glucose to an amino acid end product; and, producing anamino acid end product from the granular starch slurry.
 2. The processaccording to claim 1 wherein the contacting comprises simultaneouslyhydrolyzing the granular starch in a reaction mixture comprising themicroorganism.
 3. The process according to claim 1 wherein the granularstarch substrate is obtained from corn, wheat, barley, or rice.
 4. Theprocess according to claim 3 wherein the granular starch substrate iscorn starch obtained from whole grain.
 5. The process according to claim3 wherein the granular starch substrate is dry milled or wet milled. 6.The process according to claim 3 wherein the dry solids content is15%-55%.
 7. The process according to claim 3 wherein the contacting stepis conducted at a pH of 5.0 to 6.0.
 8. The process according to claim 3wherein the alpha amylase is derived from a Bacillus.